Transcript Lecture II
Lecture II 5- collection of fraction and visualization Fraction could be collected based on 1- fixed volume 2- specified time Similar compound are bulked together Detection of the bands could be On column detection - coloured compounds - Using UV Out of column detection -Fractions are collected and subjected to UV light - Colour reagent for fraction or its TLC Factors affecting column efficiency 1- Column dimensions 2- Adsorbent 3- Mobile phase 4- Sample loading 5- Temperature Applications of column chromatography 1- purification of natural compounds isolated from the plants 2- separation of end products in organic synthesis 3- sample preparation for other sophisticated equipment (HPLC, GC) 4- preparative scale of wide array of natural products of plants origin e.g. alkaloids, flavonoids, cardiac glycosides II- Planar chromatography A- Thin layer chromatography It is the simplest chromatographic technique TLC involves: a- selection of stationary phase b- selection of mobile phase c- sample loading d- layer development e- visualization f- identification of the isolated compounds g- preparative TLC h- Applications of TLC Selection of stationary phase and layer preparation -particle size (1-25 um are recommended for TLC -binder are often included - stationary phases are available as bulk powder Cellulose /a lu lic a Si Polar inorganic Polar organic al co ar Ch m in a Stationary phase ce ll u los e non Polar inorganic ac eta te non Polar organic Application of some selected adsorbents 1- Silica gel: alkaloids, amino acids, steroids 2- Mg silicate mainly for lipid separation 3- Alumina: alkaloids, steroids, carotene 4- Polyamide; mainly or flavonoids 5- Cellulose powders; for alkaloids, amino acids and food dyes Binder These are substances incorporated with the adsorbent to help binding the adsorbent to the plate and to produce relatively durable abrasion resistant layer E.g. Gypsum (CaSO4), silicon dioxide, starch Home made TLC a- adsorbent, wetting solvent, glass or plastic foil b- preparation of the layer Silica + distilled water. shack to give slurry then spread over glass plate.........dry at room temp. Then activation by heat at 110-120 c for 1-2hrs 4- Pre-coated TLC These are commercial ready to use plates, available in different thickness 1-2 mm for analytical and preparative Advantages of pre-coated TLC - do not need activation - ready RF is guaranteed 5- Modified silica Silica is subjected to pre-treatment to be used for separation of certain substances - CuSO4 (amines) DMF (tetrahydrocannabinol) AgNO3 (for compound have difference in number , position of =) C- Selection of mobile phase Depend on solute character D- Sample application a- Manual by capillary b- Automatic applicator Sample is applied as spot or line at 1.5 cm distance away from the lower edge and 1cm away from side edge E- Forms of plate development Development in TLC is a term describing the process in which the developing solvent runs through the sorbing layer thereby spreading the sample In column - linear mode is only possible In TLC/PC Linear, radial, horizontal TLC Linear Single Radial Horizontal Continuous Multiple Two dimension One dimension Isocratic Gradient A- linear regular development ascending movement solvent B- multiple development -to obtain better separation of complex mixtures. the development can be performed either with one solvent system in several runs or with different systems, the plate (chromatogram) should be dried after each development process. to increase the resolution & the accuracy of the separation process. C- continuous development In this case solute with low RF value can migrate to the top D- Two dimensional TLC The development is carried on a rectangular sheet, the sample is spotted to one of the corners & the 2′nd development is carried out at right angle to the direction of the first run, development maybe performed either with identical systems in both directions, or with 2 different systems, this method is used for the separation of complex substances. E- horizontal run F- visualization of chromatogram Visualization universal biological specific enzymatic detection irreversible formation of coloured derivative destructive non destructive use of corrosive UV material as H2SO4 water spray for saponin Some visualizing agents Aniline phthalate...........reducing sugars Dragendorff`s..................alkaloids FeCl3.....................phenolic compounds Ninhydrin...................amino acid, amino sugars Anisaldehyde H2SO4......terpene, sterols, sugars Identification of the separated substances By comparison of RF with authentic sample RF= distance travelled by solute/ Distance travelled by solvent Factors affecting RF value 1- choice of adsorbent 2- choice of mobile phase 3- volume of sample to be spotted 4- temperature Preparative TLC ( PTLC) A- layer and sample Layer thickness increase (double) Application of sample in form of bands with or without possible multiple development Operating condition the same as TLC B- Visualization of PTLC Non destructive method - UV light For non uv absorping compounds there are several alternative method - water spray method - edge spray method C- recovery of the sample -Sample is scraped off plate by the spatula -Solute is extracted with the least volume of solvent (acetone or ethanol or chloroform) - Filter Applications of TLC 1- detection and monitoring compound through a separation process 2-qualitative and initial screening of plant extract 3- in field of natural products separation -opium alkaloids, cannabis, ipeca, cinchona, glycosides,............................ 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