thin layer chromatography theory

7/29/2019 Thin Layer Chromatography Theory

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Thin Layer Chromatography (TLC)

O

HN+

O O-

N+

O O-

OH

2-nit rophenol 4-nit rophenol

O

HN+

O O- N+

O O-

OH

2 -n it ro ph en o l 4 -n it ro ph en ol

In the case of the mixture of two compounds, the interaction with the silica gel willbe stronger for 4-nitrophenol: cannot H-bond within itself.

Mobile phase: solvent

Polar solvents will compete with the stationary phase due to their high affinityto the polar compoundsAs a result, compounds will move faster in polar solvents

However, the order of elution will be the same

Hexanes methylene chloride ethyl acetate

IncreasingPolarity of solventsHexanes

TolueneMethylene chloride

Dietyl etherEthyl acetateAcetone

Methanol

Water

Retardation factor

The Rf(=retardation factor) depends on

solvent system

absorbent (grain size, water content,thickness)amount of material spottedtemperature

Rf= distance compound traveleddistance solvent traveled

Thin Layer Chromatography (TLC)


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Visualization techniques

Sulfuric acid/heat: destructive, leaves charred blots behind

Ceric stain: destructive, leaves a dark blue blot behind for polar compoundsIodine: semi-destructive, iodine absorbs onto the spots, not permanentUV light: non-destructive, long wavelength (background green, spots dark),

short wavelength (plate dark, compounds glow), Do not look into the UV lamp!!!

Circle the spots on the TLC plate to have a permanent record how far the

compound traveled on the plate. Also draw a sketch of the developed plate inyour lab notebook.

http://www.analtech.com/mit-video-on-thin-layer-chromatography.html

Troubleshooting TLC

Problem:The plate solvent front runs crookedly.

Reason: Either the adsorbent has flaked off the sides of the

plate or the sides of the plate are touching the sides of the

container (or the paper used to saturate the container) as the plate

develops. Crookedly run plates make it harder to measure Rf

values accurately.

Problem: Many random spots are seen on the plate.

Reason: Make sure that you do not accidentally drop any organic compound on the

plate. If get a TLC plate and leave it laying on your workbench as you do the

experiment, you might drop or splash an organic compound on the plate.

Problem:No spots are seen on the plate.

Reason:Solvent level is above baseline.

Troubleshooting TLC

Problem: The compound runs as a streak rather than a spot

Reason: The sample was overloaded.

Solution: Run the TLC again after diluting your sample. Or, your

sample might just contain many components, creating many spots

which run together and appear as a streak. Perhaps, the experiment

did not go as well as expected.

Problem: The sample runs as a smear or a upward crescent.

Reason: Compounds which possess strongly acidic or basic

groups (amines or carboxylic acids) sometimes show up on a

TLC plate with this behavior.

Solution: Add a few drops of ammonium hydroxide (amines) or

acetic acid (carboxylic acids) to the eluting solvent to obtain

clearer plates.

Preparative TLCLarger samples (up to 100 mg) can be separated

Sample is applied as a band not a spot

Baseline is drawn 1.5 inch above the edge

The components are isolated as bands

The plate is dried, the bands are traced with a pencil, scraped, and sonicated with a

polar solvent

Silica gel is gravity filtered, and the solution is rotary evaporated to afford purefractions


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Monitoring the reaction progress

10 min 20 min 40 min 1hour

H2NNH2 NH

HN O

OCH3

OCH3

O

F3C

NH

HN O

OCH3

OCH3

O

H3CO

H3COa

b, c

12 13

+

14

. , -

Analyzing the reaction mixture

H2NNH2 NH

HN O

OCH3

OCH3

O

F3C

NH

HN O

OCH3

OCH3

O

H3CO

H3COa

b, c

12 13

+

14

. , -

Determining the purity of the product

H2NNH2 NH

HN O

OCH3

OCH3

O

F3C

NH

HN O

OCH3

OCH3

O

H3CO

H3COa

b, c

12 13

+

14

. , -

A P A P

pt m zat on o t e con t ons or separat on y

column chromatography

Hexanes 70:30Hex/EtOAc 50:50Hex/EtOAc 30:70Hex/EtOAc

For the best results, choose solvent mixtures in which different components

will have Rf0.3Start with the least polar solventRealize that everything will be moving much faster on the column: so start

with at least 20% lower polarity

Ethyl acetate

thin layer chromatography theory

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