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Chromatography

Maria Salvacion A. Esmalla, M. Sc.
Biochemistry Department
Faculty of Pharmacy
University of Santo Tomas

Objectives
• To separate the components of Capsicum frutescens / Moringa
oleifera
• To perform column chromatography and thin layer chromatography
• To identify the colored pigments present in Capsicum frutescens /
Moringa oleifera
• To determine the polarity of the colored pigments
• To compute the Rf value of the colored pigments

Plant Sources
• Capsicum frutescens

• Moringa oleifera

What are the pigments present in plants? How can we separate the pigments? .

separation is based on the rates at which the components of a mixture are carried through a stationary phase by a mobile phase .Chromatography separation of mixtures due to differences in the equilibrium distribution of sample components between stationary phase and mobile phase.

Stationary vs Mobile Phase Stationary Phase – It is fixed in place. either in a column or a planar surface Mobile Phase – Moves over or though the stationary phase carrying the pigments in it .

and van der Waals forces Silica and Alumina the most commonly used adsorbents in liquid chromatography are silica (SiO2) and alumina (Al2O3) . hydrogen bonding. dipole-dipole.Stationary Phase: Adsorbent porous or finely divided solids (or liquids) in which a thin layer of molecules of some substances can be adhered organic compounds interact with the adsorbents through the following types of interactions: ion-dipole. dipole induced dipole.

Mobile Phase: Eluent is a solvent or solvent system used to carry the components of a mixture through a stationary phase polarity of the eluent/solvent affects the relative rates at which compounds move through the column ELUTION .

Chromatogram pattern formed by substances that have been separated by chromatography output or technique result of the chromatographic .

least polar Reverse Phase chromatography .mobile phase: least polar or non-polar higher Rf.mobile phase: polar higher Rf.stationary phase: least polar or non-polar .Classification: Polarity of the Stationary and Mobile Phase Normal Phase chromatography . most polar .stationary phase: polar .

ascending (through capillary action) 2.Types of Chromatography Based on direction of movement of the solvent 1. descending (due to action of gravity) Based on concentration of the solvent system 1. Gradient . Isocratic 2.

TLC Partition chromatography .particles are separated on the components of the solvent system .Classification: Mechanisms of Separation Adsorption chromatography .particles are adsorbed onto the surface of a polar packing (solid support) .the stationary phase is the surface of a finely divided polar solid . paper chromatography . column chromatography.a solute distributes between two immiscible phases (gas-liquid or liquid-liquid) .e.e.g.g.

Classification of Chromatographic Methods Column Chromatography MOBILE PHASE Gas Liquid STATIONARY PHASE TYPE Solid. immobilized Affinity (hydrogen bonding) biosorbent Liquid Liquid-liquid (partition) . gel Gel permeation (steric exclusion) Solid. molecular sieves Gas-solid Liquid Gas-liquid (partition) Solid. ion exchanger Ion exchanger (ion exchange) Solid. adsorbent Gas-solid (adsorption) Solid. adsorbent Liquid-Solid (adsorption) Solid.

thin layer Thin layer (adsorption) .Classification of Chromatographic Methods Planar Chromatography MOBILE PHASE Liquid STATIONARY PHASE TYPE Paper Paper (partition) Solid.

Sample Preparation .

Column Chromatography one of the most useful methods for the separation and purification of solids and liquids when purifying small quantities of material adsorbent the stationary phase is a powdered adsorbent packed into a column uses polarity differences to separate components of mixture cotton plug .

Column Chromatography Elution with the Solvent System solvent the sample is subjected to two opposing forces: sample mixture adsorbent (1) the solubility of the sample in the elution solvent system (2) the adsorption forces binding the sample to the solid phase .

Column Chromatography Development of Chromatogram ©Mr. Salcedo . A. R. M.

Salcedo .Theoretical Elution Order Alkanes Alkenes Ethers Increasing polarity Halogenated hydrocarbons Aromatic hydrocarbons (move more slowly) Aldehydes and ketones Esters Alcohols Amines Carboxylic acids ©Mr. M. R. A.

DCM:hexane (1:1) 2.Column Chromatography Components of Capsicum frutescens Stationary phase: silica gel Mobile phase: (Eluents) 1. DCM 3. DCM: methanol (1:1) .

Column Chromatography Components of Capsicum frutescens .

Column Chromatography Components of Capsicum frutescens CAPSANTHIN (red) CAPSORBIN (red-orange) .

Column Chromatography Components of Capsicum Frutescens ß-CAROTENE (yellow) .

Column Chromatography Components of Capsicum frutescens Fatty acid ester of capsanthin and capsorbin – contributes primarily to the deep red shade of Capsicum frutescens ß-CAROTENE – responsible for the yellow pigment of Capsicum frutescens .

Hexane:acetone (7:3) 2. Acetone 3.Column Chromatography Components of Moringa oleifera Stationary phase: silica gel Mobile phase: 1. Acetone: methanol (1:1) .

Column Chromatography Components of Moringa oleifera lutein zeaxanthine .

Column Chromatography Components of Moringa oleifera Chlorophyll a (dark green) Chlorophyll b (lime green) O .

Column Chromatography Components of Moringa oleifera Phaeophytin a & b (dark gray and bluish gray) .

stationary phase used is in the form of thin layer.Thin Layer Chromatography . either a sheet of paper or a slurry of the adsorbent and water solvent front adsorbent spots of sample origin .

To monitor column chromatography. 5. To determine the number of components in a mixture. 2.Thin Layer Chromatography Some common uses of TLC 1. To determine the appropriate conditions for a column chromatographic separation. To determine the identity of two substances. To monitor the progress of a reaction. 6. 4. 3.To determine the effectiveness of a purification. .

Evaluation .Thin Layer Chromatography Steps: 1. Visualization 5. Equilibration 3. Sample Loading/ Application 2. Development of Chromatogram 4.

Thin Layer Chromatography Sample application the sample is applied as a concentrated spot with small dimension significant loss in resolution is a consequence of applying too large amount of the sample .

Thin Layer Chromatography Development of Chromatogram .differential partitioning occurs between the components of the mixture dissolved in the solvent and the stationary adsorbent phase .solvent rises by capillary action up through the adsorbent .

UV light: non-destructive. spots dark). not permanent 4. Ceric stain: destructive. Iodine: semi-destructive. leaves a dark blue blot behind for polar compounds 3. compounds glow) . short wavelength (background green.Thin Layer Chromatography Visualization Techniques 1. iodine absorbs onto the spots. Sulfuric acid/heat: destructive. long wavelength (plate dark. leaves charred blots behind 2.

Thin Layer Chromatography Documentation and Interpretation of Data Rf = Distance from origin to center of substance spot Distance from origin to solvent front .

it depends on the following parameters: solvent system adsorbent amount of material spotted temperature .Thin Layer Chromatography Retention factor.is characteristic for any given compound on the same stationary phase using the same mobile phase for development of the plates . Rf value .

used to detect the presence of a conjugated pi bond system in a molecule The Electromagnetic Spectrum .Ultraviolet Spectroscopy .

↓ energy ↓ ↓ .Ultraviolet Spectroscopy Observed Color of Compound Color of Light Absorbed Approximate Wavelength of Light Absorbed Green Red 700 nm Blue-green Orange-red 600 nm Violet Yellow 550 nm Red-Violet Yellow-green 530 nm Red Blue-green 500 nm Orange Blue 450 nm Yellow Violet 400 nm conjugation. wavelength. ↓ frequency.

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