Lesson 3.5. Chromatography

Chromatography

separation technique for gases, liquids and dissolved substances based on the different degrees of attractive interaction of molecules to either the mobile phase or the stationary phase.

“khromatos”

means color

graphia

means recording or description

affinity

Any kind of chromatography will separate substances based on their __________ with the mobile phase and stationary phase

Stationary Phase

• layer or coating on the supporting medium (column or planar surface) which interacts with the analytes

• contains adsorbents (ex: silica, paper)

Mobile Phase (Solvent System)

part of the chromatographic system which carries the solute across the stationary phase

Polarity, Hydrophobicity, Ionic Interactions, Hydrogen bonding, Particle size, Structural complementarity

HOW DOES CHROMATOGRAPHY WORKS?

Normal

CLASSIFICATION OF CHROMATOGRAPHY

Based on the nature of MP and SP

• Stationary phase: POLAR (ex: silica gel)

• Mobile phase: NON-POLAR (ex: 3:1 chloroform and methanol)

Reverse

CLASSIFICATION OF CHROMATOGRAPHY

Based on the nature of MP and SP

• Stationary phase: NON-POLAR (ex: n-octyl or n-octyldecyl)

• Mobile phase: POLAR (ex: methanol)

Ascending

CLASSIFICATION OF CHROMATOGRAPHY

Based on direction of movement of solvent

• capillary action of solvent

• ex: TLC chromatography

Descending

CLASSIFICATION OF CHROMATOGRAPHY

Based on direction of movement of solvent

• gravitational pull to the solvent

• ex: column chromatography

Isocratic

CLASSIFICATION OF CHROMATOGRAPHY

Based on concentration of solvent

• uses only one mobile phase

Gradient

CLASSIFICATION OF CHROMATOGRAPHY

Based on concentration of solvent

• uses different ratios of 2 or more mobile phase

Partition

CLASSIFICATION OF CHROMATOGRAPHY

Based on Mechanism of Separation

• Particles are separated on the components of the solvent system

• its affinity relies heavily on the solvent/mobile phase

• e.g. paper chromatography

Adsorption

CLASSIFICATION OF CHROMATOGRAPHY

Based on Mechanism of Separation

• Particles are adsorbed in the solid support

• the stationary phase has an effect

• e.g. silica gel chromatography, thin layer chromatography

P-anisaldehyde stain

Common TLC Stains

• The greatest advantage of this stain is that different colors are manifested on TLC on heating for different molecules. Therefore, molecules can be differentiated even if they have the same Rf values.

• The disadvantage is its strong but pleasant odor release during heating (toxic, in the hood!)

Cerium sulfate stain

Common TLC Stains:

• General stain.

• Most compounds are stained brown or yellow.

Cerium molybdate stain

Common TLC Stains:

• One of the most sensitive stains which detects most functional groups.

• The disadvantage is that everything stains blue

Iodine/I2

Common TLC Stains:

• Everything stains yellow.

• Solid subs can be added to a developing chamber and the TLC plate developed by placing the plate in the chamber.

KMnO4

Common TLC Stains:

• Detects molecules with an “oxidizable” functional group.

• Relatively insensitive, every- thing stains yellow, frequently even without heating.

Ninhydrin Solution

Common TLC Stains:

• Especially sensitive to amino acids, as well as amines and anilines.

• Avoid contact with skin.

Phosphomolybdic acid (PMA)

Common TLC Stains:

• Everything stains blue–green.

• A very sensitive stain, possibly used most often

Vanillin Stain

Common TLC Stains:

• Different colors are manifested on heating for different molecules.

aromatics and conjugated systems

UV light reacts with:

aromatics

Iodine strongly reacts with ________________

aldehydes, ketones, and alcohols

p-Anisaldehyde and vanillin are used for __________________

alkenes, alkynes, or oxidizable groups (aldehydes and alcohols)

Permanganate is for _______________

alcohols, phenols, alkenes, and carbonyl compounds

Phosphomolybdic Acid (PMA) is for _________________

phenols

Iron (III) Chloride is used for ____________________

acidic compounds

Bromocresol Green is used for ___________________

Column chromatography

another form of solid-liquid adsorption chromatography and depends on the same fundamental principles as does thin-layer chromatography

Gas-liquid chromatography (GLC)/gas chromatography (GC)

technique that may be used to separate mixtures of volatile compounds whose boiling points may differ by as little as 0.5 °C.

identify the components of a mixture

Gas-liquid chromatography (GLC)/gas chromatography (GC) can also be applied as an analytical tool to _________________________________ or in preparative applications when quantities of the pure components are desired.

mobile gaseous phase and a stationary liquid phase

Gas-liquid chromatography operates on the principle of partitioning the components of a mixture between:

carrier gas

Gas-Liquid Chromatography:

a. In practice, a sample is injected into a heated chamber where it is immediately vaporized and carried through a column by a flowing inert gas such as helium or nitrogen, which is called the ______________.

b. This _______________ is the mobile phase.

c. The ___________ is packed with a finely divided solid support that has been coated with a viscous, high-boiling liquid, which serves as the stationary phase.

a = ?

gaseous mixture

Gas-Liquid Chromatography:

a. In practice, a sample is injected into a heated chamber where it is immediately vaporized and carried through a column by a flowing inert gas such as helium or nitrogen, which is called the ______________.

b. This _______________ is the mobile phase.

c. The ___________ is packed with a finely divided solid support that has been coated with a viscous, high-boiling liquid, which serves as the stationary phase.

b = ?

column

Gas-Liquid Chromatography:

a. In practice, a sample is injected into a heated chamber where it is immediately vaporized and carried through a column by a flowing inert gas such as helium or nitrogen, which is called the ______________.

b. This _______________ is the mobile phase.

c. The ___________ is packed with a finely divided solid support that has been coated with a viscous, high-boiling liquid, which serves as the stationary phase.

c = ?

Fourier Transform Infrared Spectroscopy (FTIR)

technique used to identify molecules by studying how they absorb infrared (IR) light