(A) 2.3 Paper Chromatography or Thin Layer Chromatography

This method of separation is used to see what coloured materials make up e.g. a food dye analysis (e.g. smarties), separating the different coloured dyes in an ink e.g. felt tip pen inks.

Chromatography can be used to identify substances and check on the purity of a substance.

For example you could then investigate whether a pen ink is made up of one or more colours by paper chromatography.

The different food colourings in confectionary products e.g. in the icing top of a cake, the sugar coating on smarties etc. can all be separated and identified using paper chromatography.

The coloured material mixture to be separated e.g. a food dye (6 on the diagrams below) is dissolved in a solvent like ethanol ('alcohol') and carefully spotted onto chromatography paper or a thin layer of a white mineral material on a glass sheet (immobile or stationary phase). 

Alongside it are spotted known colours of pure dyes on the reference 'start line' (1-5), which is drawn in pencil so it doesn't 'run or smudge' - see the diagram below.

The paper is carefully dipped into the solvent (mobile phase) and suspended so the start line (baseline) is above the liquid solvent, otherwise all the spots would dissolve in the solvent!

The solvent is absorbed into the paper and rises up it as it soaks into the paper.

The solvent may be water (aqueous solvent) or an organic liquid non-aqueous solvent like an alcohol (e.g. ethanol, butanol) or a hydrocarbon (e.g. hexane).

The technique and principles of how to do paper chromatography is illustrated below.

A DIAGRAM TO ILLUSTRATE the principles of PAPER CHROMATOGRAPHY

(a) At the start mark a baseline in pencil - this mark won't dissolve or run. Make sure it is above the surface of the solvent, so as not to dissolve the spots directly. You then carefully put spots of the mixtures and standards (known materials) onto the baseline. so we start with an even line of solute chemical spots e.g. dyes or any coloured compound. The prepared chromatogram paper is carefully placed in the solvent so the baseline and spots are above the solvent - and the solvent (grey) begins to move up the paper.	(b) As the solvent seeps into the paper and moves (spreads) up the paper it dissolves the ink spots and the colours begin to separate out. It is best done in a larger covered beaker so the solvent doesn't evaporate into the laboratory!	(c) When the solvent is near the top of the paper, the paper is removed and allowed to dry before examination and measurement of the Rf values of all the dyes. The final result is called the chromatogram. If any of the dyes are insoluble in the solvent, they just stay on the start line and no analysis of them can be done. The 'finish line' is the highest point reached by the solvent front.	(d) How do we measure an Rf value from the resulting chromatogram? Its quite simple to measure a reference value ratio (Rf) for a dye colour. e.g. for the green dye spot, S might be 7.2 cm, D might be 5.3 cm, so Rf = D/S = 5.3/7.2 = 0.74

For best results:

(i) try different solvents eg water or ethanol ('alcohol'), propanone ('acetone') or butanol.

(ii) If you can enclose the whole system in a large glass container with a lid on.

This reduces evaporation of the solvent.

(iii) The fastest moving spots should be able to move at least 5 cm to get a reasonably accurate R_f value.

(iv) However, if any of the dyes are insoluble in the solvent, they just stay on the start line and no analysis of them can be done because there is no separation to make an R_f measurement (see below) to compare with known standard dye colours.

For accurate work the distance moved by the solvent is marked on carefully with a pencil and the distances moved by each 'centre' of the coloured spots is also measured.

These can be compared with known substances BUT if so, the identical paper and solvent must be used (See R_f values below and diagrams above).

The R_f values is the ratio of how far the spot travels (D on diagram) relative to the distance moved by the solvent front (S on the diagram) and must have values of >0 and <1 to be of any use.

Each dye has its own reference values R_f for a particular set of experimental conditions

Chromatography R_f vales depend on the substance, the type of paper (stationary phase) AND the solvent (mobile phase).

To get consistent chromatography results you must use identical conditions, including temperature, and identical chemical reagents. These criteria is an example of 'fair testing' methodology.

You may have to experiment with solvents to ensure the best separation of the components for the most accurate analysis.

Generally speaking if spots match up, it shows what is present, if no matching spot, that dye wasn't present in the mixture.

However, (i) sometimes you get a match in one solvent and (ii) not another - this means (i) is a coincidence, because the reference dye and component mixture dye spots should match up whatever the component - need to think carefully about this point.

 

THEORY of thin layer/paper chromatography e.g. for a series of dyes

In chromatography the separation of the components in a mixture depends on their distribution between the mobile phase and the stationary phase.

Due to different solubility's of the coloured chemicals in the solvent AND different strength's of molecular 'adhesion' attraction to the paper, some colours move more than others up the paper faster, so effecting the separation of the different coloured molecules.

So the effectiveness of the separation depends on how soluble the chemical is in the solvent and how strongly the chemical is attracted to the paper.

Different solvents would be tested to see which was the most effective in separating the materials.

The distance the substance travels up the paper depends on:

(i) How soluble the substance is in the solvent - the mobile phase.

(ii) How strongly attracted the substance is to the paper - the stationary (immobile) phase - this is an intermolecular attraction.

Since these both differ from substance to substance, a separation takes place, the most soluble in the solvent or the least strongly attracted to the paper, will travel the farthest up the paper to give the largest R_f value.

Each solute dye is distributed between the paper and the solvent (the two phases) and there is constant movement of molecules between the stationary and mobile phases.

At any instant there is a dynamic equilibrium of dye concentration between the phases, meaning the number of dye molecules moving from the paper to the solvent equals those moving from the solvent to the paper.

Each dye has a different solubility and different strength of attraction to the paper - its all about interaction with the mobile solvent phase (ease of dissolving) and intermolecular attractive forces between the paper and the dye molecules.

However, if one dye is more strongly held by the paper (stationary phase), and the less soluble it is in the mobile phase solvent,  its progress up the paper is slowed down, hence the separation of the colours.

The final result is the vertical separation of the spots up the paper which is now referred to as the chromatogram.

Any colour which horizontally matches another is likely to be the same molecule i.e. red (1 and 6), brown (3 and 6) and blue (4 and 6) match, showing these three coloured substances are all in the food dye (6), the dye that's being analysed by paper chromatography.

In the diagram, think of dye (colouring) spots 1 to 5 as the known food dye colours, therefore food colouring dye 6 must be a mixture of three dyes, that is food colourings 1, 3 and 4 because these are the spots that line up horizontally with known standard samples. Dye 6 is composed of a mixture of red (1), brown (3) and blue (4) dyes.

Note that 1. to 5. give one spot each AND that's what you would expect for a pure compound. In fact it would be useless to have impure standards!

Suppose the solvent reached 8.0 cm from the baseline and for the coloured substances e.g

red spot 1.5 cm, R_f = 1.5/8.0 = 0.19;  green spot 4.5 cm,  R_f = 4.5/8.0 = 0.56;  blue spot 7.5 cm,  R_f = 7.5/8.0 = 0.94

The distance a substance moves, compared to the distance the solvent front moves (top of grey area on 2nd diagram) is called the reference or R_f value and has a value of 0.0 (not moved, not soluble, no good), to 1.0 (too soluble - no good either), but R_f ratio values between 0.1 and 0.9 can be useful for analysis and identification of coloured dye-like materials.

R_f = distance moved by dissolved substance (solute) / distance moved by solvent front

Rf values are used in many analytical situations to identify substances e.g. forensic science - analysing evidence samples, dye analysis in the food industry, food additives, testing for drugs, biochemistry e.g. analysing amino acids in a protein structure

Some technical terms: The substances (solutes) to be analysed must dissolve in the solvent, which is called the mobile phase because it moves. The paper or thin layer of material on which the separation takes place is called the stationary or immobile phase because it doesn't move.

It is possible to analyse colourless mixture of chemicals if the 'spots' can be made coloured by some further chemical or light treatment e.g.

(i) protein can be broken down into amino acids, separated with paper chromatography and coloured purple by a chemical reagent called Ninhydrin.

Each protein has its unique Rf value for a particular solvent.

(ii) Many colourless organic molecules fluoresce when ultra-violet light is shone on them, so the spots show up under uv light.

These extra reagents are called locating agents and enable R_f values to be measured and amino acids and lots of molecules to be identified.

If a substance is pure, only one spot will appear on the chromatogram, impurities may show up as other faint spots.

Thin layer chromatography (t.l.c, in chemistry, is not 'tender loving care'!) is where a layer of paste is thinly and evenly spread on e.g. a glass plate. The paste consists of the solid immobile phase like aluminium oxide dispersed in a liquid such as water (thick paste and dried out) or silica gel. The mobile phase or solvent is just the same as paper chromatography e.g. ethanol, other alcohol etc.

Important note: If the starting spot moves up the paper and remains as a single spot, it means that substance must be pure and not a mixture. If it was a mixture, then at least two spots would be seen.

Gas-liquid chromatography is further down this page.

For more on the chromatography of amino acids see:

GCSE notes AMINO ACIDS and natural polymers

Advanced A Level Notes Amino acids - molecular structure, preparation and reactions

A plant material extraction process using chromatography

The experiment described below simulates one way in which drug companies extract plant material to develop products in the pharmaceutical industry.

1. Take some suitable plant material and crush it with a pestle and mortar.

2. Scrape the crushed plant material and gently heat with a small volume of suitable solvent like water or alcohol to dissolve and extract some of the soluble coloured plant material.

3. Filter off the residue to give a clear but coloured concentrated solution.

4. Take the solution and spread it along the start line of some chromatography paper (diagram A).

5. Dip and suspend the chromatography paper into a suitable solvent in a covered container so the start line is above the solvent surface and let the solvent be absorbed by the chromatography paper and move upwards (diagram B).

6. When the solvent front has reached near the top of the paper, stop  (diagram C) and hang up the chromatogram to dry.

7. You can then separate the mixture 'physically' by cutting strips off for each band of coloured material that has separated out on the chromatogram (diagram D).

8. You can then extract each separated product by re-dissolving it by placing the strips in the solvent.

9. The different solutions can then be carefully evaporated to produce the separated coloured solid materials.

Note: In practice in the pharmaceutical industry large scale thin layer chromatography would be used.

Another example of how to interpret a chromatogram where paper chromatography has been used to analyse coloured materials.

Lets assume we have pure red, blue and yellow dye molecules.

These three rise to different heights on the chromatograms.

Three different Rf reference values (yellow > red > blue).

By matching the spots horizontally, you can deduce:

(i) The purple ink contains red and blue dye molecules.

(ii) The green ink contains blue and yellow molecules.

(iii) The brown ink contains red, blue and yellow molecules.