PLANAR CHROMATOGRAPHY

Submitted byS. Srilakshmi

M.Pharm. 1st Yearyy14mph328

Department Of Pharmaceuticals Analysis

Under the guidance ofMs. Srujani M.Pharm., PhD.

ACHARYA NAGARJUNA UNIVERSITY COLLEGE OF PHARMACEUTICAL SCIENCES

Chromatography Chromatography is a technique for separating

mixtures into their components in order to analyze, identify, purify, and/or quantify the mixture or components.

Separate• Analyze

• Identify

• Purify

• QuantifyComponentsMixture

Mechanisms Of Separation

Partitioning equilibrium ( SP: liquid, MP: liquid/gas )

Adsorption equilibrium ( SP: solid, MP: liquid )

Exclusion equilibrium ( SP&MP: liquid )

Ion Exchange equilibrium ( SP: solid ion-exchanger, MP:

liquid electrolyte )

Affinity equilibrium ( SP: immobilised ligand, MP: liquid)

Classification Of Chromatography

chromatography

Liquid chromatography Gas chromatography

Flat chromatography Column chromatography

Paperchromatography

Thin layer chromatography

(TLC)

Ascending Two dimensional CircularDescending

High performanceLiquid

chromatography

Open column( e.g.-Partition

chromatographyAdsorption

chromatographyIon exchange

chromatographyGel filtration

chromatographyAffinity

chromatography )

ClassificationColumn Chromatography

The stationary phase is held in a narrow tube through which the mobile phase is forced under pressure or by gravity.

Planar Chromatography

The stationary phase is supported on a flat plate or the interstices of a paper and the mobile phase moves through the stationary phase by capillary action or by gravity.

Planar Chromatography - Types

Thin layer chromatography (TLC)

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).

Paper Chromatography (PC)

Separates dried liquid samples with a liquid solvent (mobile phase) and a paper strip (stationary phase).

THIN LAYER CHROMATOGRAP

HY (TLC)

Thin layer chromatography (TLC)

In TLC, any substance that can be finely divided and formed into a uniform layer can be used.

Both organic and inorganic substances can be used to form a uniform layer for TLC.

Organic substances include: cellulose, polyamide, polyethylene.

Inorganic: silica gel, aluminum oxide and magnesium silicate.

Continue…

TLC to separate lipids

Surface of the plate - very thin layer silica – SP

Silica – polar (stationary phase)

Spot the material at the bottom of the TLC plate

cellulose Al2O3

Continue…

Place the plate into a glass jar - small amount of a solvent

This solvent - moving phase.

Remove the plate from the bottle when the solvent is close to the top of the plate.

Thin-Layer Chromatography: A Two-Component Mixture

More polar!

Less polar!

solvent frontorigin mixture

solvent front

component B

component A

origin

solvent front

component B

component A

origin

Increasing Development Time

Thin Layer Chromatography

TLC plate

O O O | | | O Si O Si O Si O H

| | | O O O | | | O Si O Si O Si O H

| | | O O O

silica gel - silicon dioxide (SiO2)x

(a common, inexpensive stationary phase)

bulk (SiO2)x

These exposed OH unitsgive silica gel a

relatively polar surface.

surface

Four Stages in TLC

1. Sample Application - Capillary used to spot solution of each sample.

2. Development - This is when the separation actually occurs.

3. Visualization - viewed under UV light.

4. Interpretation of Result - Comparison of retention factors.

TLC plate

“finishing line” 1 cm.A. Draw “guide lines”

lightly with pencil

“starting line” 1 cm.

B. Dissolve solid sample in MeOH

C. Use TLC capillary to transfer and spot dissolved sample

Sample A B C Ref. Ref. Ref.

1. Sample Application (spotting)

2. Development of TLC Plate

TLC plate

TLC Developing Chamber (just a glass jar with solvent in it!)

A. Place spotted TLC plate in developing chamberB. Developing solution is drawn up the plate by capillary action

C. Remove TLC plate when when solvent reaches top line

Developingsolution

(mobile phase)

}

{keep capped}

NOTE: During this ~20 min.developing stage, compoundsin the original spots are beingpulled through the silica gel.

3. Visualization of TLC Results

A. Allow solvent to evaporate from surface of TLC plate.

C. Mark spots with a pencil while viewing under UV.

UVB. View results under UV light. look for grayish spots on the fluorescent green background

4. Interpretation of TLC Results

A. Determine retention factors (Rf) for each spot detected.

B. Use Rf’s of reference spots to identify the other components.

distance spot has moveddistance solvent has moved

_______________________Rf = = XY

Y

- - - - - - - - - - - - - - - - - - - - - - -

X3

- - - - - - - - - - - - - - - - - - - - - - - - - - -

- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -

X1

X2

How do you interpretany other spots?

4

1

3

2

??

Thin Layer Chromatography- Applications

1. Separation of carbohydrates:

Mobile phase: acetonitrile : water (85:15)

Detection: sulfuric acid : methanol (1:3)

Heat for 10 min at 110 C to see brown spots

Separation of Total Lipid into different Classes

Mobile Phase: hexane: diethyl ether: formic acid (80:20:2)

Cholesteryl esters

TAG

Free fatty acids

Cholesterol

1,3-DAG

1,2-DAG

Monoacyl glycerols

Phospholipids

Separation of Triacylglycerols

Mobile Phase: Pet ether: diethyl ether: acetic acid (90:9:1)

Tristearin

2-oleodistearin

1-stereodiolein

Triolein

Trolinolein

With HUFA

Paper Chromatography Purpose

Use the technique of paper chromatography to separate a homogeneous mixture into its individual components.

Uses Separation Identification

Chromatography paper

Stationary phase

Solvent

Mobile phase

Paper Chromatography

Continue… Paper chromatography is a variant of partition

chromatography procedure in which the cellulose support is in the form of a sheet or paper.

Cellulose contain a large amount of bound water even when extensively dried.

Partitioning occurs between the bound water and the developing solvent.

Continue…

In paper chromatography the mixture to be separated is spotted onto the paper and dried

Then the solvent flows along the sheet either by gravity ( descending chromatography ) or capillary attraction (ascending chromatography )

Place 25 mL of solvent in a 600 mL beaker. Cover the beaker and set it aside.

25 mL

1 cm

2 mm

Obtain a piece of chromatographypaper and draw a line 1 cm from thebottom with a pencil.

Place a small spot of each indicator on the line.

Procedure

Spot and label each of the four indicators and one of the unknowns.

The spots should be about 2 cm apart.

2 cm

When the spots have dried, re-spot each one.

Continue…

When the spots have dried, form the paper into a cylinder with the spots facing out. Staple the edges together being careful to keep them straight and not allowing them to touch.

Place the cylinder into the 600 mL beaker and replace the cover. Be sure the cylinder is not touching the sides of the beaker.

Continue…

Let the chromatogram develop until the solvent is 2 cm from the top of the paper.

Remove the chromatogram from the beaker and immediately mark the solvent front with a pencil.

Allow the chromatogram to dry before going to the next step.

Continue…

Take the chromatogram to the hood and lightly mist it with water. Place it in the ammonia chamber.

Remove the cylinder from the ammonia chamber and unroll it. Immediately circle the colored regions with a pencil.

Continue…

Continue…

Determine the RF values for each colored spot in the known and the unknown.

ab

cdRRF(a)F(a) = = aa

dd

Use your computed RF values to identify the components of your unknown.

Paper Chromatography-Applications

Separation of amino acids

Mobile phase: butanol : acetic acid: water(4:1:1) Detection: spray with ninhydrin reagent

Separation of carbohydrates:

Mobile phase: ethylacetate : pyridine water(10:4:3) Detection: 1. silver nitrate (1 ml in 200 ml of acetone) 2. 40% NaOH in methanol gives brown

spots