Chromatography

By Sunil (+918437550953) Lovely professional University, Phagwara ,Punjab ,India

Sunil Kumar ( Msc. Botany)

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
Analytical - determine chemical composition of a sample

Preparative - 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 patients blood stream

Hospital detect blood or alcohol levels in a

Law Enforcement to compare a sample found at
a crime scene to samples from suspects pollutants in the water supply

Environmental Agency determine the level of

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 Chromatography

Gas - solid

Gas - 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. 2. 3. 4. Adsorption Ion Exchange Partition 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 cant 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

Types of Chromatography
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) samples with a liquid solvent (mobile phase) and a paper strip (stationary phase) samples with a liquid solvent (mobile phase) and a glass plate covered with a thin layer of alumina or silica gel (stationary phase)

Paper Chromatography separates dried liquid

Thin-Layer Chromatography separates dried liquid

STATIONARY PHASE
Type of chromatography Material
Paper chromatography (KK = kertas kromatografi) Thin Layer Chromatography (KLN = Kromatografi lapisan nipis) Gas chromatography (GC) High Performance Liquid Chromatography (KCPT = kromatografi cecair prestasi tinggi) Filter paper, cellulose Silica gel, alumina, polyamide Squalene, apezion, carbowax M C-8, C-18, Licosorb, Silicone

MOBILE PHASE
Type of chromatography Solvent Paper chromatography Air, alcohol (KK = kertas kromatografi) Thin Layer Chromatography (KLN = Kromatografi lapisan nipis) Hexane, ether petroleum, alcohol.

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

He, Ar, N2 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 mixture ex: water, alcohol. With capillary action the solvent will move up to filter paper. Movement of a solvent will bring together component that are separated from the mixture. Every component that are separated will move to several velocity

The moving components are depend on : a. Solubility solute in solvent b. Intermolecule forces c. Pore size of filter paper d. 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:

Materials List
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

Preparing the solvent solution

Prepare the solvent solution in various concentration: - 0%, 5%, 10%, 20%, 50%, and 100%

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 covered 4. 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