Topic outline● Description of Planer Chromatography● Description of theoretical principles of Planer Chromatography● Degree of retention (Rf)● ApplicationsDr. Khalid Hussain (University College of Pharmacy) firstname.lastname@example.org
Planer Chromatography● This chromatography is performed on plane surface, rather than a column● It offers a unique advantage of 2-Dimentional operation● Selective properties of 2 different solvents can be used in developing a single chromatogram● It includes: paper chromatography (PC) and thin layer chromatography (TLC)/High performance thin layer chromatography (HPTLC)
Theoretical principles● The principles of column chromatography also apply here i.e.1- Separation is accomplished by successive equilibrations of the sample components between two phases, the mobile phase (m) and stationary phase (s).2- Non-ideal process, similar to column chromatography, cause zone spreading on the plane● Degree of retention in plane chromatography is expressed as retardation factor (Rf)
Rf = Distance solute moved Distance solvent moved For substances that are very soluble in the liquid, Rf will be close to .... 1 For substances that are rather insoluble in the liquid, Rf will be close to .... 0 Hence, Rf lies between 0 and 1 Solvent Front Line Distance traveled by solvent Distance traveled by spot Origin LineGoogle images
Distance of the solute moved is measured from thecentre of the spot In column chromatography, distribution coefficient is expressed by K=Cs/CM
The simple relationship between K and Rf is given as follows:Rf = Number of moles of solute in moving phase / Number of moles of solute in both phases Rf= CmAm/ CmAm+CsAsHere Am and As are the cross-sectional areas of the two phasesDividing numerator and denominator by CM, we get Am Rf = Am+CsAs/Cm Since, K= Cs/CM = Am/Am+KAs
Factors effecting Rf● Rf values are subject to minor influences such as1- Variations of stationary phase2- Method of application and development3- Size of sample4- Concentration of sample
Paper Chromatography● It is mainly used for qualitative and semi- quantitative purposes● It is easy to perform● Mechanisms involved: liquid-liquid partition, adsorption, hydrogen bonding, and ion exchangeNature of the paper● Made up of highly purified cellulose- a polar compound- hence has great affinity for water and polar solvents, hold them through H-bonding● Paper may be impregnated with alumina, silica and ion exchange resin etc.
Procedure● Application of sample as a small spot● Development in a closed chamber saturated with solvent either by1- Ascending method:● Simplest and most popular● Solvent ascends through the paper by capillary action● The rate of ascent is slow and decrease with the passage of time due to gravity● Slow rate enhances possibility of achieving partition equilibrium. Google images
2- Descending method:● Direction of flow of solvent is downward● Paper is folded U-shape to prevent rapid siphoning of solvent● Solvent descends through gravity and capillary action● It is much faster than the ascending method Google images
Detection● After the development, solute may be detected by a number of methods briefed as: – Inherent visible colors – Derivitization – UV absorbance, fluorescence – IR absorbance – Radioactivity – Chemical tests – Bio-autographySinensetine 2
Applications● Qualitative (measurement of Rf value)● Isolation● Purification● Semi-quantitative1-Extract the compound from the paper followed my spectrophotometry2- Densitometric determination.
Techniques● Two dimensional development● Radial developmentSample is spotted at the centre and developed, components move radially forming circles of increasing diameters. Fast separation by moving disc so solvent move by CF and CA● Reversed-phase chromatography.Paper is coated with hydrophobic substance, polar phase is used to elute, separation of non-polar compounds
Thin Layer ChromatographyVery popular method and often used same as pc. Thin layer offinely divided adsorbent is supported on glass or plastic plates.It can be used for qualitative, quantitative and preparativepurposes.1- Here the mobile phase is a liquid, flowing past a thin layer of powderon a solid support2- Substances that are less attracted to the solid or more soluble in liquidmove faster.3- And so move further up the plate by the time that the process has beenstopped by taking the plate out of the liquid- larger Rf
Nature of the layer● Commonly used adsorbents are silica gel, alumina, diatomaceous earth and powdered cellulose.● Silica gel which is acidic and has high capacity, is useful for adsorption and partition chromatography● Alumina is basic and used primarily for adsorption chromatography● Diatomaceous earth is nearly neutral and used for partition chromatography● Thickness of the layer varies from 0.15-2.00 mm● TLC plates can be prepared in Lab or purchased commercially (much more convenient and more reproducible than home-made plates)
Procedure of development ● Same as paper chromatography, mostly By ascending method or horizontal methodIsocratic GradientCamag website
3. Detection:If the spots are not colored and can’t be seen by the eye, use:• UV lamp for UV-active compounds (most aromatics are UV-active)• If compounds are not UV-active, use derivatizationOnce you visualize the spots, circle them with a pencil.4. Calculate Rf values for each spotRf = distance spot traveled from origin line/distance of solventfrontRf values will help to identify an “unknown”Make sure to use the same mobile phase as Rf’s will varywith varying mobile phases.
Modern TLC● Automatic (sample application, development, detection and quantification)● Advantages1- Cheap and easy2- More versatile than pc3- Faster and more reproducible4- Often used for complex sample separation and visualization5- Modern TLC complementary to HPTLC4- Many samples and standards can be applied on the same plate