Thin Layer Chromatography (TLC)

H3CO O H2N NH2 H N H3CO

Thin Layer Chromatography (TLC)

TLC is a simple, effective, and inexpensive method for: monitoring the progress of the reaction analyzing the reaction mixture identifying the components of the mixture determining their purity optimizing the solvent system for a given separation problem Advantages: Requires small amounts of the compound Very fast technique

a b, c

F3C

N H

O OCH3 OCH3

N H

H N

O OCH3 OCH3

12

13

14

a) 1 eq ethyl triflouroacetate; b) 1.1 eq 2, 3-dimethoxybenzoyl chloride; c) excess Et3N; d) 2 HCl; e) 1 eq NaOCH3

Questions you should ask: When do I stop the reaction: it cannot tell me! How do I know if I made the product? What if I am the first one to make this product? How do I know if it makes any sense to isolate and purify the product: I do not want to waste my time! How do I choose the conditions for product purification: I have my product, and I do not want to mess up on purification step! All these questions can be answered by using a simple TLC technique

Principle of separation
Components of the mixture are separated based on their afnity for the stationary and mobile phase Stationary phase: silica gel, alumina coated on glass or plastic strip Mobile phase: solvents

Stationary phase
Silica gel is a polymeric form of silicic acid The presence of hydroxyl groups makes the silica gel very polar
HO Si O HO Si O OH Si O OH Si O OH Si O O H

Si

When a polar compound is applied to a TLC plate, it will form a H-bond interaction with the polar silica hydroxy groups.

H NO2 O H H O Si O HO Si O OH Si O OH Si O OH Si O O H O O N+ O-

Si

Thin Layer Chromatography (TLC)

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

Mobile phase: solvent

Polar solvents will compete with the stationary phase due to their high affinity to the polar compounds As a result, compounds will move faster in polar solvents However, the order of elution will be the same Hexanes Toluene Methylene chloride Dietyl ether Ethyl acetate Acetone Methanol Water

O N+ OO H N+ OO 4-nitrophenol

IncreasingPolarity of solvents

2-nitrophenol

O N+ OO H O 2-nitrophenol N+ O-

Hexanes

methylene chloride

ethyl acetate

4-nitrophenol

Retardation factor
Rf = distance compound traveled distance solvent traveled The Rf (=retardation factor) depends on solvent system absorbent (grain size, water content, thickness) amount of material spotted temperature

Thin Layer Chromatography (TLC)

Visualization techniques
Sulfuric acid/heat: destructive, leaves charred blots behind Ceric stain: destructive, leaves a dark blue blot behind for polar compounds Iodine: semi-destructive, iodine absorbs onto the spots, not permanent UV 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 in your lab notebook.

Troubleshooting TLC
Problem:The plate solvent front runs crookedly. Reason: Either the adsorbent has aked 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.
http://www.analtech.com/mit-video-on-thin-layer-chromatography.html

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.

Larger 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 ltered, and the solution is rotary evaporated to afford pure fractions

Preparative TLC

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.

Monitoring the reaction progress

H3CO O H2N NH2 H N H3CO

Analyzing the reaction mixture

H3CO O H N O OCH3 OCH3 H2N NH2 H N H3CO

a b, c

F3C

N H

O OCH3 OCH3

a b, c

F3C

N H

N H

O OCH3 OCH3

N H

H N

O OCH3 OCH3

12

13

14

12

13

14

a) 1 eq ethyl triflouroacetate; b) 1.1 eq 2, 3-dimethoxybenzoyl chloride; c) excess Et3N; d) 2 HCl; e) 1 eq NaOCH3

a) 1 eq ethyl triflouroacetate; b) 1.1 eq 2, 3-dimethoxybenzoyl chloride; c) excess Et3N; d) 2 HCl; e) 1 eq NaOCH3

10 min

20 min

40 min

1hour

Determining the purity of the product

H3CO O H2N NH2 H N H3CO

Optimization of the conditions for separation by column chromatography

H N

a b, c

F3C

N H

O OCH3 OCH3

N H

O OCH3 OCH3

12

13

14

For the best results, choose solvent mixtures in which different components will have Rf 0.3 Start with the least polar solvent Realize that everything will be moving much faster on the column: so start with at least 20% lower polarity

a) 1 eq ethyl triflouroacetate; b) 1.1 eq 2, 3-dimethoxybenzoyl chloride; c) excess Et3N; d) 2 HCl; e) 1 eq NaOCH3

Hexanes

70:30Hex/EtOAc

50:50Hex/EtOAc

30:70Hex/EtOAc

Ethyl acetate