chromatography-1.pptx chromatography and

Chromatography

1. Chromatography

2. Introduction to chromatography 1) Derived from Greek word Chromatography Chroma means “ color” and graphy means “ to write ” . 2) Micheal tswett was the discoverer of chromatography. He was a botanist . 3) He Discovered Chromatography by experimenting on plants

3. Chromatography Experiment Took leaves of a plant, grind them well, colorless extract separate out, used Chromatography technique and obtained 3 bands. Chlorophyll Green Caratenoid Red Xanthophyl Yellow

4. Chromatography Technique used to separate complex mixtures present in solution into separate colorfull bands is called Chromatography. This technique based on two phases Mobile phase Stationary phase

5. Chromatography Mobile phase Moving phase in Chromatography is called mobile phase. can be liquid and gas. cannot be a solid Stationary phase Non-moving phase/static phase or phase on which stationary phase move is called stationary phase. Can be solid or liquid, cannot be a gas

6. On Base of Mechanism 1) Adsorption Chromatography( adsorption+absorption phenomena) 2) Ion exchange Chromatography( on base of attraction to resin ) 3) Partition Chromatography ( solubility phenomena) 4) Size exclusion/Gelpermeation( On size of solute )

7. On Base of Mobile phase Liquid Chromatography Gas Chromatography Super critical fluid (CO 2 is used as a gas)

8. On Base of Stationary phase Solid chromatography Liquid chromatoraphy

9. On Base of Chromatographic tank 1) Planner type ( paper , TLC) 2) Column type ( ion exchange) Importance of chromaatography 1) Separation of mixtures 2) Qualitative analysis 3) Identification of compounds 4) Medical labs 5) Purification

10. Paper Chromatography separate the components in a mixture type of partition chromatography (SP is liq) liquid-liquid chromatography Filter paper available On base of porosities (small,medium,large) On base of shape (circular, rectangular)

11. Types Ascending type Descending type Radial type

12. Thin Layer Chromatography Thin plate ( glass, Al) commonly used slurry is silica,alumina,cellulose Make a thin layer on glass plate, that thin layer act as SP Small amount of MP used ( different solvents) SP( polar) MP(non polar) MP move by capillary action against gravity

13. TLC SP is of two types 1) inorganic ( silica,alumina, MgO, Ca and Mg silicates) 2) Organic ( cellulose,charcoal)

14. Preparation of TLC plate heat glass plate infurnace at 1100oC for 3 - 4min make sulrry ( 100g silica in 200-250ml of water), make thick paste 4 methods used to deposit slurry on plate 1) Pouring 2) Dipping 3) spraying(liq slurry) [by atomizer] 4) spreading( just like bread on slice, best method bcoz even slurry spread) 500-2000micrometer thickness of slurry Now heat slurry at 1000 for 30min

15. Qualities of MP Should be easily available should be less expensive should be volatile should be inert to separating components

16. Visualizing agents Iodine chamber Put TLC plate in iodine chamber, brown colored spots formed mostly) 10% H2SO4 in ethanol reacts with almost all compounds and give black color. Cobalt Chloride ( universal stain) Ferric chloride FeCl3 ( for phenol) gives red color Ninhydrin (for amino acids)

17. Column Chromatography Different components of a mixture separate with help of column Column size used is mostly 1:10 SP ( silica,alumina,MgO) Use cotton plugs SP is also called adsorbent

18. Column Chromatography Types of columns 1) should be inert ( mostly glass, stainless steel) 2)available in different sizes and design 3)for closely related components( large long size used) 4)for different compounds small sized used. 5)We use acid washed silica

19. Gravity Column Simple glass column in which MP move in direction of gravity) . Flash Column glass column in which N2 or air is passed with pressure to increase the flow rate of MP . Low pressure colum MP is pumped through using a pump and pressure is kept low . Vacuum colum By using vacuum pump,create low pressure at end , MP will move from upward high Pressure to downward low Pressure High pressure Column Finely divided SP used to increase resolution ,due to which rate of flow of MP become slow. High pressure is used to increase flow rate.

20. Packing Of Column cotton plug use . Acid washed sand used Two types Wet packing make slurry( sand + water thick paste),and fill in column while vibrating for homegenize packing.preferred over dry. Dry packing dry or powder silica used as such. More cracks or air bubbles formed,resolution not good.

21. Characteristics of SP should be 60-200 u m should be inert inexpensive easily available should allow free movement of MP should be cololess particles of SP should be spherical