Thin-Layer Chromatography

Carlos Kruut
September 11, 2024

Experiment Overview:
Reagents & Solvents:

Reagent Table:
Name Hexanes Ethyl Fluorene Fluorenone Benzoic Acid
Acetate

Structure

Molar Mass 86.178 g/mol 88.11 g/mol 166.223 180.192 122.12 g/mol
g/mol g/mol
Density 0.6606 g/mL 0.9006 1.202 g/mL 1.13 g/mL 1.27 g/mL
g/mL
Amount Used 10 mL 1 mg 20 mg 20 mg 20 mg
Concentration
Notes Reference Reference
Safety Safety

Safety:
• Appropriate lab attire such as gloves and goggles must be worn during this experiment.
• Ethyl acetate must be handled carefully avoiding inhalation and skin exposure. This liquid
is highly flammable, can cause drowsiness and dizziness if inhaled, and can cause eye
injury if in contact with ethyl acetate.
 • Hexanes are made of about 60% n-hexane, various amounts of 2-methylpentane and 3-
methylpentane, and small amounts of non-hexane-isomer alkanes. Hexanes can cause eye,
skin, and respiratory irritation, drowsiness and dizziness if inhaled, and organ damage and
death if swallowed. The use of a chemical fume hood with this solvent is paramount.
• UV light is harmful to the retinas when looked at directly so avoid contact from the light
source.
Waste:
• TLC solvents must be disposed of in the organic waste container.
• TLC spotters must be disposed of in the broken glass waste.
• TLC plates must be disposed of in a solid waste container.
Glassware and Equipment:
• TLC developing chamber and lid
• 2 TLC plates
• 8 vials
• TLC spotter (either capillary tube or melting point tube)
• Forceps
• UV Lamp
Terms & Techniques:
• When drawing the lines on the 2 TLC plates, apply light pressure with the pencil and create
a solid line across.
• Capillary tubes suck liquid up via capillary action which is intermolecular forces between
the solid surface and the liquid surface. To use a capillary tube for this experiment, plug
one end when submerged in the liquid, hold over the TLC plate, and touch gently to the
corresponding plate letter.
Spot Distance (from middle of spot to baseline)
• Calculating Rf is as follows: 𝑅𝑓 = Total Distance (solvent front to baseline)

Protocol:
Preparation of Eluents
• Get TLC developing chamber and lid.
• Solvent 1: 100% hexanes Each solvent = 1 of 4 lab partners
• Solvent 2: 30% ethyl acetate and
70% hexanes Each student needs to make 5 mL of their
• Solvent 3: 70% ethyl acetate and solvent.
30% hexanes
• Solvent 4: 100% ethyl acetate Solvent = eluent
• Put eluent in TLC developing chamber and Make sure the lid is closed to prevent
close lid. evaporation and allow for headspace
permeation.
• Get 2 TLC plates and draw straight line Handle TLC plates on the sides (edges) to
with pencil on each along silica side 1 cm prevent flaking.
from bottom. Silica side is the non-shiny side.
 From the line down is the baseline.
• Draw four small vertical lines along A = Fluorene Standard
horizontal line (equally spaced) and label B = Fluorenone Standard
the lines A, B, C, and D under the vertical C = Benzoic Acid Standard
lines. D = Compound Mixture
• Obtain and label 4 vials for the group Label vials A,B,C, and D
• Add to the vials: 20 mg of fluorene to A, 20 Each lab partner will add to their
mg of fluorenone to B, 20 mg of benzoic respective vial.
acid to C, and 20 mg of compound mixture
to D.
TLC Plate Preparation
• Add 1 mL of ethyl acetate to each vial, cap, Goal is to use as little ethyl acetate as
and shake gently to fully dissolve. possible to dissolve. Add dropwise if not
dissolved.
• Obtain TLC spotter. Spotter may be a capillary tube (open) or
melting point tube (open and closed).
• Use spotter to draw up each A-D and tap Smaller dot = clearer results
onto corresponding line intersection. Clean spotter for each standard/mixture
using acetone
• Label vial “Acetone” and fill with some Empty spotter by tapping onto paper
wash acetone to use to clean spotter. towel, dip in acetone, tap onto paper towel.
Repeat this twice per mixture and let dry
for 30 s between each sample.
• Once plate is prepared, use forceps to place Make sure baseline is above solvent
plate in developing chamber with baseline initially.
in first. Minimize contact between plate and
chamber wall.
• Recap chamber While holding base in place
Solvent Front and Rf
• Allow plate to run until solvent front is 1 Solvent front is the solvent line moving up
cm from top of plate. the plate
• Remove plate from chamber with forceps
and immediately draw line horizontally
where solvent front is.
• Dispose chamber solvent in organic waste Chamber solvent → organic waste
• Remove lid and allow solvent to evaporate
• After 2 minutes of solvent evaporating off Do not look into UV
plate, see sample spots under UV lamp and
outline using a pencil.
• Calculate Rf value for each spot using Spot 1:

Spot 2:
Spot Distance(from middle of spot to baseline)
Rf= Total Distance(solvent front to baseline) Spot 3:
 Spot 4:

Determination of Greatest Overall Separation

• Compare with lab partners and see which 2 Greatest 2 separations of the solvent
created the largest separation (∆𝑅𝑓 ) system:
• Create 2 new solvent systems with a Solvent system ratios picked:
polarity in between the 2 largest solvent
systems. (stick to whole # such as 40/60, 50/50)
• Using the procedure above make a new Make sure they each go up to 5 mL of the
TLC chamber using new solvent systems. solvent system.
• Solvent 1: Original choice 1 Solvent 1:
• Solvent 2: Original choice 2 Solvent 2:
• Solvent 3: Intermediate choice 1 Solvent 3:
• Solvent 4: Intermediate choice 2 Solvent 4:
• Repeat procedure with 2nd TLC plate and Greatest overall separation of the 4:
determine greatest separation.
Waste Disposal
• Dispose of waste as discussed in the waste
disposal section.

Results: