Experiment 4: Chromatography

An ubiquitous method of analyzing and purifying organic compounds which is based on polarity differences between molecules to be separated in a mixture

What is chromatography?

List 4 basic separation and purifications techniques

1. Recrystallization
2. Distillation
3. Chromatography
4. Extraction

Which purification technique is based on solubility?

What is Recrystallization?

Which purification technique is based on polarity differences?

What is Chromatography?

Which purification technique is based on boiling point?

What is Distillation?

Which purification technique is based on pH values and/or solubility differences based on compound polarity

What is Extraction?

What does chromatography involve?

laboratory techniques for the separation of mixtures involving passing a mixture which contains the compounds to be separated dissolved in a mobile phase, through a stationary phase which separates them based on differences of absorption to the stationary

Chromatography is the ___________ of a mixture into its __________ .

physical separation
individual components

What is liquid chromatography used for?
What is gas chromatography used for?

To identify unknown plant pigments & other components
To determine the chemical comp of unknown substances such as compounds in gasoline.

Which type of chromatography uses thin plastic or glass trays to id the comp of pigments, chemicals and other unknown substances

Thin-Layer chromatography

Every type of chromatography involves what?

The distribution of a substance between two phases, a mobile phase and a stationary phase. The mobile phase is an organic solvent and the stationary phase, a polar surface.

True or False: The more polar a substance, the more it will move along with the mobile phase?

False! A particular molecule will partition between being bound to the surface (stationary phase) where it doesn't move, and being dissolved in the eluent (mobile phase) such that it flows along. The more polar, the more a molecule will bind to the more p

In TLC chromatography, what is the plastic or glass plate coated with?

In TLC chromatography, what is the plastic or glass plate coated with?

How does the flow of the eluent travel up the TLC plate?

by capillary action.

In TLC chromatography, what determines how far up the plate, the sample will travel?

The mobility of a particular chemical depends on its partition between the mobile phase (the eluent) and the stationary phase (silica gel). The more tightly the sample binds to the silica, the less it will move. The less well it binds, the more it will di

How do you develop a TLC plate?

1. In a jar or beaker, line inside with filter paper which will serve to increase solvent saturation, add solvent to under 1 cm (below spots on tlc plate) and soak filter paper, cover with watch glass
2. Spot TLC plate w/ sample using capillary tube (1 cm

T or F: The solvent used to Develop the TLC plate should be at a level above the sample spots.

False, if the solvent is above the spots, the sample spots will dissolve into the pool of solvent instead of travel up the plate.

How can you visualize spots that are not visible? (clear sample)

1. Stain with Iodine
2. UV light Detection

How does staining with Iodine work?

This is a non destructive visualizing technique. A few crystals of iodine are placed in a closed chamber, such as a capped jar containing silica gel, the slide is placed into the chamber and by weak electronic attraction, iodine will collect on the spots

Is the Iodine stain reversible?

Yes, if allowed to sit for a few minutes the iodine will sublime off the spots.

How does UV light detection work?

Another non destructive visualizing technique that shows any compound that absorbs UV light as a dark spot on the commercially treated plate which fluoresces when exposed to long-wave UV. (compounds such as benzene rings and conjugated systems will show u

List some destructive visualizing techniques and breafly describe:

1. Permanganate stain - Any oxidizable compound will show up as a brown-yellow spot. (MnO2)
2. PMA stain (phosphomolybdic acid stain) - fairly universal stain. Some amines, amides and oxidation-resistant aromatics won't show up. Stain solution is somewhat

What is the Rf value? How is it calculated?

The retention factor, is the distance traveled by the compound / distance traveled by solvent.

What are some important uses of TLC

1. estabilished that two compounds are identical
2. determines # of compounds in a mixture
3. determines appropriate solvent to use in column chromatography
4. used to monitor column chromatographic separation
5. check effectiveness of separation using va

What happens if the solvent used is too polar?

A solvent which is too polar will result in all the spots running to the top of the plate, the Rf value will be zero, this is indicative of a bad solvent for separation

What happens if the solvent used isn't polar enough?

The spots will not move from the base line and the Rf value will be zero this is not a good solvent for separation

What is a good Rf value for a vsolvent?

A good Rf solvent will give an Rf value of 0.3 to 0.7 for the desired compound and atleast a difference of 0.1 between the compound and impurities.

How does Column Chromatography work?

Through the same principle as TLC. through differential intermolecular forces between the components of the mixture with the mobile phase, and the stationary phase. A variety of adsorbents can be used as the stationary phase, very polar silica gel, is com

Which alumina (basic, acidic, or neutral) would you use to purify carboxylic acid?

Acidic Alumina

Which type of Alumina would you use to separate neutral products?

Neutral alumina

Which type of Alumina would you use to separate amines?

Basic alumina

Packing Column: What are the two ways in which the adsorbent is packed in the column? Describe each

1. Slurry packing (Wet method) - The adsorbent is suspended in the mobile phase and stirred very well to drive off all the air bubbles. The slurry is then poured in the column. Before application of slurry a glass wool or cotton must be applied to prvent

Loading sample on column: What are the two ways to load the sample on the column? Describe each

1. Wet application - Dissolve sample in small amount of initial mobile phase and apply by long pipette to the top of the column. Drain eluent from column until no sample appears above the sand. Use 1 mL of eluent to rinse container and pipet and transfer

True or False: When eluting the sample, begin with the most polar solvent and using force pipet it directly into the sand causing a slurry to form!

False, 1st start with the least polar solvent and carefully pipet an inch or two of solvent at the top of the column in order to avoid disturbing the sand.

When developing a column, what do you call it when the same composition of the mobile phase is used from beginning to end of separation?

An isocratic system

What is it called when the polarity of the system is increased gradually during the separation by increasing the proportion of the more polar solvent. (start with hexane, then follow with hexane/ethyl acetate, finish with just ethyl acetate for example)

A Gradient

Which solvent is more polar?
1. Cyclohexane or Water
2. Carbon tetrachloride or ethanol?

1. Water
2. Ethanol

When viewing a TLC plate spotted with a mixture of A and B, compound A traveled further up the TLC plate than B, when doing column chromatography to separate the two, which will elute first? Which is more polar?

A will elute first, it traveled farthest because it was the least polar and stuck to the polar silica plate the least.
B is the more polar and will elute last.

What is the purpose of adding sand before the stationary phase in column chromatography?

The purpose of the sand at the bottom of the column is so that there is a level silica gel line since the bottom of the column is typically cone shaped, if no sand were present, then molecules traveling down the center of the column would encounter less s

What is the purpose of adding sand after the stationary phase in column chromatography?

Sand at the top of the column aids in even loading of the sample. Sample diffuses evenly throughout the sand and onto the silica evenly. If there was no sand, the sample would stick directly where it was added and not evenly.