Chromatography (paper chromatography and tlc)

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Paper Chromatography

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Thin Layer

Chromatography

1. Paper Chromatography

Paper chromatography is a technique that involves placing a small dot of sample solution onto a strip of chromatography paper.

Adsorbent: More polar substances bond with the cellulose paper more quickly.

matrix = cellulose paper (polar substance)

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Dr Gihan Gawish

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1. Paper Chromatography

1. The paper is placed in a jar containing a

solvent such as ethanol or water then

sealed.

2. A small concentrated spot of solution that

contains the sample of the solute is applied

to a strip of chromatography paper about 2

cm away from the base of the plate

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1. Paper Chromatography-

Procedure

4. As the solvent rises through the paper, it

meets the sample mixture which starts to

travel up the paper with the solvent.

5. Paper chromatography takes from several

minutes to several hours.

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1. Paper Chromatography-

Procedure

Different compounds in the sample

mixture travel at different rates due to

1. differences in solubility in the solvent

2. differences in their attraction to the fibers in

the paper.

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1. Paper Chromatography

In this method, the solvent moves upward

against gravitational force.

The only force that cause the motion is

capillary force. So the speed of the process is

slow.

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Paper Chromatography

1.1 Ascending

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In this method, the solvent is kept in a trough at the top of the chamber and is allowed to flow down the paper.

The liquid moves down by capillary action as well as by the gravitational force.

In this case, the flow is more rapid as compared to the ascending method.

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Paper Chromatography

1.2 Descending

Because of this rapid speed, the chromatography

is completed in a comparatively shorter time.

The developing solvent is placed in a trough at

the top which is usually made up of an inert

material.

The paper is then suspended in the solvent.

Substances that cannot be separated by

ascending method, can be separated by the

above descending method.

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Paper Chromatography

1.2 Descending

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After development, the spots corresponding to

different compounds may be located by:

1. their color,

2. ultraviolet light,

3. ninhydrin

4. or by treatment with iodine vapors.

The paper remaining after the experiment is

known as the Chromatogram.

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Paper Chromatography

Analysis

Rƒ value (Retention Factor)

Rƒ =

If Rƒ value of a solution is zero, the solute

remains in the stationary phase and thus it is

immobile.

If Rƒ value = 1 then the solute has no affinity for

the stationary phase and travels with the solvent

front.

the ratio of the distance traveled by the substance

the distance traveled by the solvent.

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The final chromatogram can be

compared with other known

mixture chromatograms to

identify sample mixes, using the

Rf value in an experiment .

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Rƒ value (Retention Factor)

2. Thin layer chromatography

(TLC)

TLC is a widely used.

Stationary phase solid+ adsorbent

Adsorbent like silica gel (polar), alumina (aluminium oxide), or cellulose.

Traveling of solvent and elute via capillary action

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Silica is a typical adsorbent. It has silanol (Si-OH)

groups on its surface, which are slightly acidic, and can

interact with polar functional groups of the analyte or

eluent.

In general, an eluent with a polarity comparable to that

of the most polar analyte in the mixture is chosen.

Thus, alcohols would be selected if the analytes

contained hydroxyl groups,

acetone or esters would be selected for analytes

containing carbonyl groups, and

hydrocarbons such as hexane, heptane and toluene for

analytes that are predominantly non-polar.

Mixtures of solvents are commonly used in the context

of gradient elution.

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Plate preparation

1. TLC plates are made by mixing the adsorbent

+ small amount of inert binder calcium sulfate

(gypsum) + water.

2. This mixture is spread as a thick slurry on an

unreactive carrier sheet, usually glass ,thick

aluminum foil, or plastic (support)

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2. Thin layer chromatography (TLC)-

Procedure

3. The resultant plate is dried and activated by

heating in an oven for thirty minutes at 110 °C.

4. A small spot of sample is applied to a plate,

about 1 cm from the base.

5. The plate is then dipped in to a suitable

solvent ,such as hexane or ethyl acetate ,and

placed in a sealed container.

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2. Thin layer chromatography (TLC)-

Procedure

6. The solvent (mobile phase) moves up the plate

by capillary action and meets the sample

mixture, which is dissolved and is carried up

the plate by the solvent.

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2. Thin layer chromatography (TLC)-

Procedure

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2. Thin layer chromatography (TLC)-

Analysis

The scan of TLC plate (silica gel G) with 10 essential oils developed with mobile

phase toluene - ethyl acetate (93:7 v/v), next sprayed with vanillin in H2SO4 and

heated. From left to right oils from: bergamot, cedar, eucalyptus, syzygium,

lavandula, mint, orange, pine, spruce. Identified components: B1 and L1 - linalol,

B2 and L2 - linalyl acetate, E1 - cinneol, G1 - eugenol, G2 - carryophyllene.

Doubtfully identified components - C1 - cedrol, M3 - menthol, P1 - limonene..

A. Some samples are colorless, it can be

visualize by:

1. Manganese-activated zinc silicate

(fluorescent compound( is added to the

adsorbent

2. Iodine vapors

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2. Thin layer chromatography (TLC)-

Analysis

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IODINE has been used as a colour reagent for paper chromatography of amino-

acids and their derivatives1,2, imidazoles3, and other nitrogen-containing

compounds1,4. Steroids are also revealed by this technique, which is sufficiently

sensitive5. The method has not gained wide acceptance possibly only because it

cannot be relied on to yield a colour diagnostic of a class of compounds—some

nitrogen-containing compounds are not revealed—but its application is simple,

and if unsuccessful, other colour reagents can be applied to the same

chromatogram. In the most convenient procedure, the dried paper is exposed to

iodine vapour (supplied by a few iodine crystals in a closed battery jar) for 10–

30 sec.; or the paper may be sprayed with a solution of iodine in carbon

tetrachloride3, or may be dipped into a saturated solution of iodine in

petroleum ether (b.p. 60°–80°)5. Compounds are revealed usually as brown

spots, which fade rapidly.

3. Specific color reagents are sprayed onto the

plate (ninhydrin)

4. In the case of lipids, the chromatogram may be

transferred to a PVDF (Polyvinylidene

fluoride) membrane and then subjected to

further analysis like mass spectrometry

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2. Thin layer chromatography (TLC)-

Analysis

B. Calculate the Rf value

These values depend on

1. the solvent

2. the type of TLC plate.

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2. Thin layer chromatography (TLC)-

Analysis

It is faster runs than paper,

It has better separations,

Wide choice between different adsorbents.

It has better resolution and to allow for quantitation

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2. Thin layer chromatography (TLC)-

Advantages

1. identifying compounds present in a given

substance

2. determination of the pigments a plant contains

3. detection of pesticides or insecticides in food

4. analyzing the dye composition of fibers in

forensics

5. monitoring organic reactions

6. Assaying the radiochemical purity of

radiopharmaceuticals

1. identification of medicinal plants and their

constituents [

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2. Thin layer chromatography (TLC)-

Applications

Hydroxylapatite chromatography

Crystalline hydroxylapatite (Ca10(PO4)6(OH)2) is an

adsorbent used to separate mixtures of proteins or

nucleic acids. One of the most important applications of

hydroxylapatite chromatography is the separation of

single-stranded DNA from double-stranded DNA.

Both forms of DNA bind at low phosphate buffer

concentrations but as the buffer concentration is

increased single-stranded DNA is selectively desorbed.

As the buffer concentration is increased further, double-

stranded DNA is released. This behaviour is exploited in

the technique of Cot analysis (Section 5.3.4). The affinity

of doublestranded DNA for hydroxylapatite is so high

that it can be selectively removed from RNA and

proteins in cell extracts by use of this type of

chromatography.

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