Orgo Lab Exam
Experiment 2: Thin-layer chromatography Analysis of Components in Common
Pain-Killers
Introduction:
● Objective:
○ Identify an unknown compound by comparing its Rf values to standards
(acetaminophen, aspirin, caffeine) using Thin Layer Chromatography
(TLC).
● TLC:
○A technique to separate and analyze components in a mixture based on
their interaction with a stationary phase (silica gel) and a mobile phase
(solvent).
● UV Light:
○ Used to visualize samples on the TLC plate; dark spots indicate where
compounds absorb UV light.
● Retention Factor (Rf):
○ Rf = Distance traveled by spot / Distance traveled by solvent front
■ More polar compounds interact more with the stationary phase →
travel less (lower Rf).
■ Less polar compounds interact more with the mobile phase →
travel further (higher Rf).
● Mobile Phase:
○ Ethyl acetate/acetic acid (200:1 ratio); used to move the compounds up the TLC
plate through capillary action.
○ Criteria for a suitable mobile phase: polarity, solubility, volatility, and ability to
separate components.
○ For good separation, the mobile phase should be as non-polar as possible
● Stationary Phase:
○ Silica gel (polar), interacts with polar compounds, dipole-dipole
○ Polar molecules STICK well
○ So something more polar will adhere to silica gel, move slower
○ Compounds that interact weakly will elute faster
● Solvent choice:
○ Ethyl acetate/acetic acid
○ Moderately polar, allowing effective separation of compounds with different
polarities.
● Chemical Structures & Rf Values:
○ Aspirin: Least polar (Rf = 0.693)
○ Acetaminophen: Moderately polar (Rf = 0.536)
○ Caffeine: Most polar (Rf = 0.155)
, ○ Unknown:
Results:
● Compare the Rf value obtained to the standard
● Rf = Distance travelled by spot/Distance travelled by solvent =d1/d2
●
Discussion:
● Para-aminophenol vs Acetaminophen:
○ Para-aminophenol is more polar, it would have a smaller Rf value
● Effect of changing solvent:
○ Ethanol (more polar): Polar compounds' Rf values decrease; non-polar
compounds' Rf values increase.
○ Toluene (non-polar): Polar compounds travel more, non-polar
compounds travel less
● Example of TLC separation (other mixture):
○ Mixture: Chlorophyll a, chlorophyll b, and carotene.
○ Sample Solvent: Hexane (non-polar).
○ Development Solvent: Hexane + ethyl acetate (balanced polarity for
separation)
● Purpose of the retention factor (Rf) in TLC, and how is it calculated
○ Ratio used to compare the relative movement of a compound on the TLC
plate. Rf values help identify compounds by comparing the migration
behavior of the unknown sample to known standards.
● What are some factors that can affect the results of a TLC experiment?
○ Choice of solvent system: The polarity of the solvent determines how far
the compounds will travel.
○ Plate quality and thickness: A thicker stationary phase may cause more
interaction with compounds, slowing their movement.
○ Sample size and concentration: Too much sample can lead to streaking,
while too little may result in weak spots.
○ Development time: Overdeveloping the plate can lead to the solvent front
running too far or uneven separation.
, ● What would happen if we used a more polar, or less polar developing solvent, how would
that affect the Rf value? Why?
○ If you increase the solvent polarity you will increase the eluting power and in turn
increase the Rf value. However, keep in mind if your solvent polarity is too high you will
not be able to separate your compounds.
○ More polar mobile phases will compete with your polar analytes for binding spots on the
silica gel, increasing the Rf value
○ Vice versa if you decrease. If the polarity is too low your compounds will not move from
the starting line.
Remember:
● Mark TLC with pencil only + handle with care they are fragile
● Use the dull side of the TLC
● perform all liquid transfer inside the fume hood
● spot sizes are reasonable / spotted twice at least
● spots spaced out properly
● spots are above solvent
● cover beaker with foil
● solvent front not far enough / went too far
● solvent front is straight
● Rf are in correct order
Experiment 2: Thin-layer chromatography Analysis of Components in Common
Pain-Killers
Introduction:
● Objective:
○ Identify an unknown compound by comparing its Rf values to standards
(acetaminophen, aspirin, caffeine) using Thin Layer Chromatography
(TLC).
● TLC:
○A technique to separate and analyze components in a mixture based on
their interaction with a stationary phase (silica gel) and a mobile phase
(solvent).
● UV Light:
○ Used to visualize samples on the TLC plate; dark spots indicate where
compounds absorb UV light.
● Retention Factor (Rf):
○ Rf = Distance traveled by spot / Distance traveled by solvent front
■ More polar compounds interact more with the stationary phase →
travel less (lower Rf).
■ Less polar compounds interact more with the mobile phase →
travel further (higher Rf).
● Mobile Phase:
○ Ethyl acetate/acetic acid (200:1 ratio); used to move the compounds up the TLC
plate through capillary action.
○ Criteria for a suitable mobile phase: polarity, solubility, volatility, and ability to
separate components.
○ For good separation, the mobile phase should be as non-polar as possible
● Stationary Phase:
○ Silica gel (polar), interacts with polar compounds, dipole-dipole
○ Polar molecules STICK well
○ So something more polar will adhere to silica gel, move slower
○ Compounds that interact weakly will elute faster
● Solvent choice:
○ Ethyl acetate/acetic acid
○ Moderately polar, allowing effective separation of compounds with different
polarities.
● Chemical Structures & Rf Values:
○ Aspirin: Least polar (Rf = 0.693)
○ Acetaminophen: Moderately polar (Rf = 0.536)
○ Caffeine: Most polar (Rf = 0.155)
, ○ Unknown:
Results:
● Compare the Rf value obtained to the standard
● Rf = Distance travelled by spot/Distance travelled by solvent =d1/d2
●
Discussion:
● Para-aminophenol vs Acetaminophen:
○ Para-aminophenol is more polar, it would have a smaller Rf value
● Effect of changing solvent:
○ Ethanol (more polar): Polar compounds' Rf values decrease; non-polar
compounds' Rf values increase.
○ Toluene (non-polar): Polar compounds travel more, non-polar
compounds travel less
● Example of TLC separation (other mixture):
○ Mixture: Chlorophyll a, chlorophyll b, and carotene.
○ Sample Solvent: Hexane (non-polar).
○ Development Solvent: Hexane + ethyl acetate (balanced polarity for
separation)
● Purpose of the retention factor (Rf) in TLC, and how is it calculated
○ Ratio used to compare the relative movement of a compound on the TLC
plate. Rf values help identify compounds by comparing the migration
behavior of the unknown sample to known standards.
● What are some factors that can affect the results of a TLC experiment?
○ Choice of solvent system: The polarity of the solvent determines how far
the compounds will travel.
○ Plate quality and thickness: A thicker stationary phase may cause more
interaction with compounds, slowing their movement.
○ Sample size and concentration: Too much sample can lead to streaking,
while too little may result in weak spots.
○ Development time: Overdeveloping the plate can lead to the solvent front
running too far or uneven separation.
, ● What would happen if we used a more polar, or less polar developing solvent, how would
that affect the Rf value? Why?
○ If you increase the solvent polarity you will increase the eluting power and in turn
increase the Rf value. However, keep in mind if your solvent polarity is too high you will
not be able to separate your compounds.
○ More polar mobile phases will compete with your polar analytes for binding spots on the
silica gel, increasing the Rf value
○ Vice versa if you decrease. If the polarity is too low your compounds will not move from
the starting line.
Remember:
● Mark TLC with pencil only + handle with care they are fragile
● Use the dull side of the TLC
● perform all liquid transfer inside the fume hood
● spot sizes are reasonable / spotted twice at least
● spots spaced out properly
● spots are above solvent
● cover beaker with foil
● solvent front not far enough / went too far
● solvent front is straight
● Rf are in correct order
