ChromatographyPn. Suryati Bt. Syafri2008

LEARNING OUTCOME

After studying this topic student should

be able to :

1. Define chromatography

2. Explain classification of chromatography technique

3. Explain type of phase in chromatography

4. Describe about paper chromatography (definition, principle, method & their application) in pharmaceutical industry.

DEFINITION

CHROMATOGRAPHY

The separation of a mixture by distribution of its components between a mobile and stationary phase over time– mobile phase = solvent– stationary phase = column

packing material

HISTORY

• Chromatography (from Greek :chromatos -- color ,

"graphein" -- to write) • 1903 Tswett - plant pigments

separated on chalk columns• 1931 Lederer & Kuhn - LC of

carotenoids• 1938 TLC and ion exchange• 1950 Reverse phase LC• 1954 Martin & Synge (Nobel Prize)• 1959 Gel permeation• 1965 instrumental LC (Waters)

Purpose of Chromatography

• AnalyticalAnalytical - determine chemical composition of a sample

• PreparativePreparative - purify and collect one or more components of a sample

Uses for Chromatography

Real-life examples of uses for chromatography:

• Pharmaceutical Company – determine amount of each chemical found in new product

• Hospital – detect blood or alcohol levels in a patient’s blood stream

• Law Enforcement – to compare a sample found at a crime scene to samples from suspects

• Environmental Agency – determine the level of pollutants in the water supply

• Manufacturing Plant – to purify a chemical needed to make a product

Classification of Methods

There are two classification schemes:– mobile phase– attractive forces

Mobile Phase

• Gas (GC)• Water (LC)• Organic solvent (LC)• Supercritical fluid

(SCFC)

Classification based on Mobile Phase

Gas ChromatographyGas Chromatography

Gas - solidGas - solid Gas - liquidGas - liquid

Stationary Phase

Classification based on Mobile Phase

Liquid chromatography (LC)

Column(gravity flow) High performance

(pressure flow)

Thin layer(adsorption)

Classification based on Attractive Forces

1. Adsorption2. Ion Exchange3. Partition4. Size Exclusion

Adsorption Chromatography

Separation based on their adsorption onto the surface of solid (stationary phase).

Normal phase-like separation – Nonpolar mobile phase

for polar non-ionic compounds

Ex; Column chromatography (CC) (K.Turus), TLC, HPLC

Partition Chromatography solute are separated based on their partition

between a liquid mobile phase and a liquid stationary phase coated on a solid support.

– Normal – analyte is nonpolar organic; stationary phase MORE polar than the mobile phase

– Reverse – analyte is polar organic; stationary phase LESS polar than the mobile phase

– Ex : TLC, Paper Chromatography

Phase 2

Phase 1

Phase 2

Phase 1

Ion Exchange Chromatography

Use ionic stationary phase– ions separated on the basis of their tendency to

displace counter ions adsorbed on stationary phase (Depends on charge, hydration, “solubility”…)

Anionic stationary phases: used for cation separation Cationic stationary phases : for anion separation for ionic compounds - Ex : CC (K.turus), HPLC

Size Exclusion Chromatography

Separation is a result of “trapping” of molecules in the pores of the packing material

• Very large molecules can’t get into the pores – unretained

• Very small molecules get hung up in to pores for a long time - most retained – longest retention time

• stationary phase is a porous matrix

• Ex: CC, HPLC

• Liquid Chromatography – separates liquid samples with a liquid solvent (mobile phase) and a column composed of solid beads (stationary phase)

• Gas Chromatography – separates vaporized samples with a carrier gas (mobile phase) and a column composed of a liquid or of solid beads (stationary phase)

• Paper Chromatography – separates dried liquid samples with a liquid solvent (mobile phase) and a paper strip (stationary phase)

• Thin-Layer Chromatography – separates dried liquid samples with a liquid solvent (mobile phase) and a glass plate covered with a thin layer of alumina or silica gel (stationary phase)

Types of Chromatography

STATIONARY PHASE

Type of chromatography

Material

Paper chromatography(KK = kertas kromatografi)

Filter paper, cellulose

Thin Layer Chromatography(KLN = Kromatografi lapisan nipis)

Silica gel, alumina, polyamide

Gas chromatography(GC)

Squalene, apezion, carbowax M

High Performance Liquid Chromatography(KCPT = kromatografi cecair prestasi tinggi)

C-8, C-18, Licosorb, Silicone

MOBILE PHASE

Type of chromatography

Solvent

Paper chromatography(KK = kertas kromatografi)

Air, alcohol

Thin Layer Chromatography(KLN = Kromatografi lapisan nipis)

Hexane, ether petroleum, alcohol.

Gas chromatography(GC)

He, Ar, N2

High Performance Liquid Chromatography(KCPT = kromatografi cecair prestasi tinggi)

Cyclohexane, n-hexane, carbon tetrachloride, ethanol, methanol, air

PAPER CHROMATOGRAPHY

DEFINITION

A chromatographic analytical separation technique for complex mixtures involving the progressive adsorption of the dissolved component onto a special grade of paper.

PRINCIPLE

• The certain solvent are used to separate a The certain solvent are used to separate a mixture ex: water, alcohol.mixture ex: water, alcohol.

• With capillary action the solvent will move With capillary action the solvent will move up to filter paper.up to filter paper.

• Movement of a solvent will bring together Movement of a solvent will bring together component that are separated from the component that are separated from the mixture.mixture.

• Every component that are separated will Every component that are separated will move to several velocitymove to several velocity

The moving components are depend on :The moving components are depend on :a. Solubility solute in solventa. Solubility solute in solventb. Intermolecule forcesb. Intermolecule forcesc. Pore size of filter paperc. Pore size of filter paperd. Size of soluted. Size of solute

At the end of process, components that are separated will emerge to different distance on filter paper.

Rf values are used to identification of each the component.

• The retention factor, or Rf, is defined as the distance traveled by the compound divided by the distance traveled by the solvent

For example, if a compound travels 2.1 cm and the solvent front travels 2.8 cm, the Rf is 0.75:

• Beakers or jars• Covers or lids • Solvent (Distilled

H2O, Isopropanol)• Graduated cylinder• Filter paper• Sampel (Different

colors of pens, plant extract)

• Pencil• Ruler• Scissors• Tape

Materials List

• Prepare the solvent solution in various concentration:

- 0%, 5%, 10%, 20%, 50%, and 100%

Preparing the solvent solution

Preparing the Chromatography Strips

1. Cut filter paper

2. Draw a line 1 cm above the bottom edge of the strip with the pencil

3. Label each strip with its corresponding solution

4. Place a spot from each pen on your starting line

Developing the Chromatograms

1. Place the strips in the beakers

2. Make sure the solution does not come above your start line

3. Keep the beakers covered4. Let strips develop until

the ascending solution front is about 2 cm from the top of the strip

5. Remove the strips and let them dry

Developing the Chromatograms

Spot Detection

- Color spot observed by naked eye- Non – color spot color reagent will

give specific colors for different compound.

Example : Ninhydrin – a.amino Iodin dalam etanol –bes

(termasuk alkaloid) AgNO3 berammonia -

Karbohidrat

USES OF PAPER CHROMATOGRAPHY

1. Separation of ink dyes- To compare ink dyes use in any company.

2. Food coloring- To differentiate coloring agent used in food product such as : M&M, Smarties dan Reese candies.

3. Botanist/herbalist - To isolate plant pigment

from root and leaves