Post on 15-Jan-2016
Lecture II
5- collection of fraction and visualizationFraction could be collected based on1- fixed volume2- specified time
Similar compound are bulked togetherDetection of the bands could be
On column detection- coloured compounds- Using UVOut of column detection-Fractions are collected and subjected to UV light- Colour reagent for fraction or its TLC
Factors affecting column efficiency1- Column dimensions2- Adsorbent3- Mobile phase4- Sample loading5- 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 chromatographyA- Thin layer chromatography
It is the simplest chromatographic technique TLC involves:a- selection of stationary phaseb- selection of mobile phasec- sample loadingd- layer developmente- visualizationf- identification of the isolated compoundsg- preparative TLCh- 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
Stationary phase
Polar inorganic
Polar organic
non Polar inorganic
Silic
a / al
umin
a
non Polar organic
Cel
lulo
se Charcoal
cellulose acetate
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 flavonoids5- Cellulose powders; for alkaloids,
amino acids and food dyes
BinderThese 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 foilb- preparation of the layerSilica + 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 characterD- Sample applicationa- Manual by capillaryb- Automatic applicatorSample 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 developmentDevelopment 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 possibleIn TLC/PCLinear, radial, horizontal
TLC
Linear Radial Horizontal
Single Continuous
Multiple
One dimensionTwo 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 developmentIn 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 specific biological
enzymatic detection
irreversible formation of coloured derivative
destructive
use of corrosive material as H2SO4
non destructive
UVwater spray for saponin
Some visualizing agents
Aniline phthalate...........reducing sugarsDragendorff`s..................alkaloidsFeCl3.....................phenolic compoundsNinhydrin...................amino acid, amino
sugarsAnisaldehyde 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 adsorbent2- choice of mobile phase3- volume of sample to be spotted4- temperature
Preparative TLC ( PTLC)
A- layer and sampleLayer 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 PTLCNon destructive method- UV lightFor non uv absorping compounds there are
several alternative method- water spray method- edge spray methodC- 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 TLC1- detection and monitoring compound
through a separation process2-qualitative and initial screening of
plant extract3- in field of natural products
separation-opium alkaloids, cannabis, ipeca,
cinchona, glycosides,............................
Thank you