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SUBJECT

EXTRACTION SEPARATION AND PURIFICATION OIL AND GREASE REMOVAL

CHROMATOGRAPHY

THIN LAYER CHROMATOGRAPHY

GAS CHROMATOGRAPHY HIGH PRESURE LIQUID CHROMATOGRAPHY

Solid Phase Extrac1on

SPE uses the affinity of solutes dissolved or suspended in a liquid (known as the mobile phase) for a solid through which the sample is passed (known as the staSonary phase) to separate a mixture into desired and undesired components.

Solid Phase Extrac1on

Solid Phase Extrac1on
Solid Phase Extrac1on
Solid Phase Extrac1on

SEPARATION AND PURIFICATION

Chromatography is a technique in which compounds to be separated are distributed between a mobile phase and a sta1onary phase. In such a system, different distribuSons based on selecSve adsorpSon give rise to separaSon. There are different types of chromatography, such as paper, thin layer, or column chromatography, each with its own strengths and weaknesses.

Chromatography

Column chromatography in chemistry is a method used to purify individual chemical compounds from mixtures of compounds. Column chromatography is one of the most useful methods for the separaSon and purificaSon of both solids and liquids when carrying out small-scale experiments. The separaSon can be liquid/solid (adsorpSon) or liquid/liquid (parSSon) in column chromatography.

Chromatography

Chromatography
Chromatography

Chromatography

Column chromatography is advantageous over most other chromatographic techniques because it can be used in both analy%cal and prepara%ve applicaSons. It can be used to determine the number of components of a mixture and as well as the separaSon and purificaSon of those components. Many compounds are not visible to the eye when dissolved in a solvent or adsorbed on a adsorbent. VisualizaSon processes make these substances visible. The used techniques for this purpose include UV lights that cause fluorescence or phosphorescence and chemical reacSons that give colored compounds.

Chromatography

The sta$onary phase or adsorbent in column chromatography is a solid. The most common staSonary phase for column chromatography is

phase or adsorbent in column chromatography is a solid. The most common staSonary phase for column
phase or adsorbent in column chromatography is a solid. The most common staSonary phase for column
phase or adsorbent in column chromatography is a solid. The most common staSonary phase for column

Chromatography

These adsorbents are sold in different mesh sizes, indicated by a number on the bo\le label: silica gel 60or silica gel 230-400are a couple of examples. This number refers to the mesh of the sieve used to size the silica, specifically, the number of holes in the mesh or sieve through which the crude silica parScle mixture is passed in the manufacturing process. If there are more holes per unit area, those holes are smaller, thus only smaller silica parScles are allowed to pass the sieve. The larger the mesh size, the smaller the adsorbent parScles are.

Chromatography

Alumina is quite sensiSve to the amount of water which is bound to it; the higher its water content, the less polar sites it has to bind organic compounds, and thus the less sSckyit is. This sSckiness or acSvity is designated as I, II, or III with I being the most acSve. Alumina comes in three forms: acidic, neutral, and basic. The neutral form of acSvity II or III, 150 mesh, is most commonly employed.

Chromatography

The mobile phase or eluent is either a pure solvent or a mixture of different solvents

Chromatography •   The mobile phase or eluent is either a pure solvent or a mixture

Chromatography

The polarity of the solvent, which is passed through the column, affects the relaSve rates at which compounds move through the column. Polar solvents can more effecSvely compete with the polar molecules of a mixture for the polar sites on the adsorbent surface and will also be\er solve the polar consStuents.

Chromatography

Consequently, a highly polar solvent will move even the highly polar molecules rapidly through the column. If a solvent is too polar, movement becomes too rapid, and li\le or no separaSon of the components of a mixture will result. On the other hand, if a solvent is not polar enough, no compounds will elute from the column.

Chromatography

Chromatography

Chromatography

Chromatography

Chromatography

Chromatography
Chromatography

Chromatography

Chromatography
Chromatography

Thin Layer Chromatography (TLC)

Thin-layer chromatography (TLC) is a chromatography technique used to separate non-volaSle mixtures. Thin-layer chromatography is performed on a sheet of glass, plas1c, or aluminium foil , which is coated with a thin layer of adsorbent material, usually silica gel, aluminium oxide , or cellulose .

Thin Layer Chromatography (TLC)

TLC can be divided into three groups, PreparaSve TLC AnalyScal TLC High performance thin layer chromatography (HPTLC)

groups , •   PreparaSve TLC •   AnalyScal TLC •   High performance thin layer

Thin Layer Chromatography (TLC)

Prepara1ve TLC uses plates with relaSvely thick coaSngs (0.5-2.0 mm). Afer separaSon on the plates, spots containing from 10-1000 mg of sample can be scraped off the plates and subjected to further analysis.

the plates, spots containing from 10-1000 mg of sample can be scraped off the plates and
the plates, spots containing from 10-1000 mg of sample can be scraped off the plates and

Thin Layer Chromatography (TLC)

Analy1cal TLC uses plates usually 20 x 20 cm in size with a 0.25 mm coaSng of parScles with diameters from 12-20 mm and are developed with the solvent front moving from 10-12 cm. These plates are commonly used for qualita1ve purposes.

and are developed with the solvent front moving from 10-12 cm. These plates are commonly used
and are developed with the solvent front moving from 10-12 cm. These plates are commonly used

Thin Layer Chromatography (TLC)

High performance thin layer chromatography (HPTLC) can produce fast, high-resoluSon analysis with quanStaSve accuracy and precision close to column HPLC. It uses thinner layers of staSonary phase and smaller sample volumes, thus reducing the loss of resoluSon due to diffusion.

uses thinner layers of staSonary phase and smaller sample volumes, thus reducing the loss of resoluSon
uses thinner layers of staSonary phase and smaller sample volumes, thus reducing the loss of resoluSon

Thin Layer Chromatography (TLC)

The equilibrium between the free ( completely dissolved in the liquid or gaseous mobile phase) and adsorbed ( stuck on the surface of the solid staSonary phase) states depends on three factors:

the polarity and size of the molecule the polarity of the staSonary phase the polarity of the solvent

Thin Layer Chromatography (TLC)

The polarity of the molecules is determined by their structures. By selecSng different staSonary and mobile phases one can change the equilibrium between the free and absorbed states. It is important to understand chromatography at this molecular level because this allows one to choose mobile and staSonary phases that will separate just about any mixture of molecules.

Thin Layer Chromatography (TLC)

The staSonary phase is typically alumina (Al2O3 . xH2O)n or silica gel (SiO2 . xH2O)n. The covalent network of these absorbents create very polar materials.

xH2O)n or silica gel (SiO2 . xH2O)n. •   The covalent network of these absorbents create

Thin Layer Chromatography (TLC)

Thin Layer Chromatography (TLC)

Thin Layer Chromatography (TLC)

Thin Layer Chromatography (TLC) Common Mobile Phases listed by Increasing Polarity.

Common Mobile Phases listed by Increasing Polarity.

Thin Layer Chromatography (TLC)

SpoGng the TLC Plate Once the sample is prepared, a spohng capillary must be used to add the sample to the plate. The spohng capillaries must be extremely small.

a spohng capillary must be used to add the sample to the plate. •   The
a spohng capillary must be used to add the sample to the plate. •   The

Thin Layer Chromatography (TLC)

Thin Layer Chromatography (TLC) TLC Plate ready to be spo\ed

TLC Plate ready to be spo\ed

Thin Layer Chromatography (TLC)

Development The bo\le is filled with a small amount of the mobile phase and capped with a cork. In addiSon, a piece of filter paper is put in the bo\le to help create an atmosphere saturated with solvent.

with a cork. In addiSon, a piece of filter paper is put in the bo\le to
with a cork. In addiSon, a piece of filter paper is put in the bo\le to

Thin Layer Chromatography (TLC)

Thin Layer Chromatography (TLC)

Thin Layer Chromatography (TLC)

Thin Layer Chromatography (TLC)
Thin Layer Chromatography (TLC)

Thin Layer Chromatography (TLC)

Visualiza1on

Some organic compounds are colored. If you are fortunate enough to be separaSng organic molecules that are colored such as dyes, inks or indicators, then visualizing the separated spots is easy. However, since most organic compounds are colourless, this first method does not always work.

Thin Layer Chromatography (TLC)

In most cases observing the separated spots by UV light works well. TLC plates normally contain a fluorescent indicator which makes the TLC plate glow green under UV light of wavelength 254 nm. Compounds that absorb UV light will quench the green fluorescence yielding dark purple or bluish spots on the plate. Simply put the plate under a UV lamp, and the compounds become visible to the naked eye.

Thin Layer Chromatography (TLC)

Thin Layer Chromatography (TLC)
Thin Layer Chromatography (TLC)

Thin Layer Chromatography (TLC)

Visualiza1on reagent DetecSon of organic compounds with spraying of reagent into TLC plate

Chromatography (TLC) Visualiza1on reagent •   DetecSon of organic compounds with spraying of reagent into TLC
Chromatography (TLC) Visualiza1on reagent •   DetecSon of organic compounds with spraying of reagent into TLC

Thin Layer Chromatography (TLC)

Visualiza1on reagent 2,4-Dinitrophenylhydrazine For detec$on of aldehydes and ketones Spray plate with soluSon of 0.4 g 2,4-DNPH in 100ml 2N hydrochloric acid, add 1ml ethanol Results: Yellow-red spots will be seen.

Thin Layer Chromatography (TLC)

Visualiza1on reagent Bromocresol green For detec$on of organic acids Dip chromatogram in a soluSon of 0.1g bromocresol green in 500ml ethanol and 5ml 0.1M NaOH Results: Acids yield yellow spots on a blue background.

Thin Layer Chromatography (TLC)

Visualiza1on reagent Vanillin / potassium hydroxide f or detecSon of amines and amino acids Phosphomolybdic acid for detecSon of Fa\y acid methyl esters, keto acids, lipids, steroids, and sulphated bile acids. KMnO4 for detecSon diol, C=C, reacSve methylene, phenol, thiol, phosphine Cerium-ammonium-molybdate, CAM – Universal Anisaldehyde For detecSon of phenols, sugars, steroids, and terpenes Iodopla1nate For detecSng alkaloids, amines, and organic nitrogen compounds.

Thin Layer Chromatography (TLC)

Rf Values

In addiSon to qualitaSve results, TLC can also provide a chromatographic measurement known as an Rf value. The Rf value is the

retardaSon factoror the raSo-to-front value expressed as a decimal fracSon .

Thin Layer Chromatography (TLC)

Rf Values EssenSally it describes the distance traveled by the individual components. If two spots travel the same distance or have the same Rf value then it might be concluded that the two components are the same molecule.

For Rf value comparisons to be valid; however, TLC plates must be run under the same exact

condi1ons.

•   For R f value comparisons to be valid; however, TLC plates must be run

THE END