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Planarchromatography
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Transcript of Planarchromatography
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
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- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
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