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

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