why do the pigments separate in paper chromatography

We extracted pigments from spinach leaves. The leaves were boiled in ethyl alcohol (95%) then the extract was put on a strip of chromatography paper and suspended in petroleum ether containing 1. 5% propyl alchohol. Different pigments moved up the paper. What are these substances and how does chromatography work? The original mixture of pigments are carried up the chromatograhy paper by the ascending stream of organic (non-polar) solvent. The (stationary) fibres of the paper contain water (a polar solvent), adsorbed from the humidity in the air. So essentially, there is a moving layer of non-polar solvent passing over a stationary layer of polar solvent. The speed at which a particular pigment moves depends on its relative affinities for the two solvent phases; if it has no affinity whatever for the water phase, it will travel at maximum speed, just behind the solvent-front (eg beta-carotene); on the other hand, if the pigment has no affinity whatever for the non-polar phase, it will remain stuck at the origin of application (eg anthocyanins). Most photosynthetic pigments such xanthophyll and alpha- and beta-chlorophyll have some portions of the molecule which have affinity for one solvent and portions with an affinity for the other. As a result these pigments travel along with parts of them "dragging" in the stationary water phase - and the greater the proportion dragging, the slower they move. Hence each pigment usually moves at a specific rate, forming discrete bands on the paper. Different cocktails of solvents can be used for paper chromatography, in order to allow the separation of different classes of substances, but in most cases, the stationary phase is water.


We think you would also find it helpful to read our TLC Worksheet. Chromatography is a method of separating mixtures by using a moving solvent on filter paper. A drop of mixture solution is spotted near one end of the paper and then dried. The end of the paper, nearest the spot, is then dipped into the solvent without submerging the spot itself. In ascending chromatography, the solvent is in a pool at the bottom and moves up by capillarity. In descending chromatography it is in a trough at the top and flows down by capillarity and gravity. The solvent flows along the paper through the spots and on, carrying the substances from the spot. Each of these will, if the solvent mixture has been well chosen, move at a different rate from the others. After a time the paper is taken out and dried: the substances can be seen at once if coloured, or located by treating with a suitable locating agent. The distance a substance travels depends upon the resultant between propelling and retarding forces. Propellors a) Solvent flow. this carries all solutes along with it and is the same for all substances. b) Solubility. this is a propelling force which tends to displace a substance from the paper and to keep it moving with the solvent. Usually the more soluble a substance is in the solvent, the more rapidly it will move along the paper. Solvents are chosen for the greatest differential solubilities of the substances concerned. Retarders a) Adsorption. this is an attraction between the cellulose of which the filter paper is made and the solutes. Adsorption is reversible and cellulose gradually releases most substances into the solvent as it flows over the spot.


Adsorption is differential like solubility: some substances are more strongly adsorbed than others. b) Partition. chromatography depends upon two non-mixing liquid phases, the solvent and the water bound to the cellulose molecules of the filter paper. When a substance which is soluble in the two non-mixing solvents is exposed simultaneously to both, it will partition itself between them. The amount found in each solvent will depend upon the relative solubility of the solute in each. The degree of partition at equilibrium is known as the partition coefficient. In fact the water forms the stationary phase and the solvent a moving phase. The water can be thought of as trapped in lots of little tubes over the tops of which the solvent is passing. When a drop is spotted on paper the solute dissolves in the water of the tubes. As the moving solvent runs over the tubes it picks up the solute by partition and redeposits some of it again by partition in succeeding tubes. As it moves, it is followed by fresh solvent and so the process repeats. As there are the equivalent of thousands of tubes, a vast number of partitions take place, so small differences in partition coefficient between different solutes of a mixture lead to good separation in the course of paper chromatography.
Paper chromatography is one method for testing the of compounds and identifying substances. Paper chromatography is a useful technique because it is relatively quick and requires only small quantities of material. Separations in paper chromatography involve the same principles as those in, as it is a type of thin layer chromatography.


In paper chromatography, substances are distributed between a stationary phase and a mobile phase. The stationary phase is the water trapped between the cellulose fibers of the paper. The mobile phase is a developing solution that travels up the stationary phase, carrying the samples with it. Components of the sample will separate readily according to how strongly they adsorb onto the stationary phase versus how readily they dissolve in the mobile phase. When a colored is placed on a filter paper, the colors separate from the sample by placing one end of the paper in a. The solvent up the paper, the various molecules in the sample according to the of the molecules and the solvent. If the sample contains more than one color, that means it must have more than one kind of molecule. Because of the different chemical structures of each kind of molecule, the chances are very high that each molecule will have at least a slightly different polarity, giving each molecule a different in the solvent. The unequal solubility causes the various color molecules to leave solution at different places as the solvent continues to move up the paper. The more soluble a molecule is, the higher it will migrate up the paper. If a chemical is very non-polar it will not dissolve at all in a very polar solvent. This is the same for a very polar chemical and a very non-polar solvent. It is very important to note that when using water (a very polar substance) as a solvent, the more polar the color, the higher it will rise on the papers.

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