diagram of paper/thin layer chromatography at the end

Mixture separation using CHROMATOGRAPHY analysis - chromatograms

Doc Brown's Chemistry KS4 science GCSE/IGCSE/O Level Chemistry Revision Notes


using Paper chromatography, thin layer chromatography and gas chromatography

PARTS 2.3 and 2.6 Methods of separating mixtures are described e.g. paper chromatography, thin layer chromatography, gc gas chromatography, glc gas-liquid chromatography

Remember, in the physical separation processes of paper chromatography and gas chromatography, no chemical reaction changes are involved, so no new substances are made.

Part 1 Definitions in Chemistry, Elements, Compounds & Mixture pictures & Physical & Chemical Changes

Part 2 Methods of Separating Mixtures of substances

Part 3 How to write equations, work out formula and name compounds

Alphabetical list of KEYWORDS for Parts 1-3: atom  *  balancing equations (work your way down the section carefully)  *  centrifuges/centrifuging  *  chemical reaction/change  *  chromatography (paper/thin layer)  *  compound  *  covalencycrystallisation  *  decanting/decantation  * displayed formula  *  distillation (simple or fractional)  *  element  *  equations  *  evaporation  *  filtration  *  formula  * gas chromatography  *  impure/pure  *  insoluble  *  ionic equations  *  ionic valence  *  iron-sulphur separation and heating experiment  *  magnet  *  mixture  *  molecule  *  naming compounds and ions  *  particle pictures of elements/compounds/mixtures  *  physical change  *  precipitation  *  products  *  pure substance  *  purification  *  reactants  *  sand/salt separation  *  separating funnel  *  separating mixtures  *  soluble/solution/solvent/solute  *  solvent extraction  *  symbols (for elements, formula, in equations)  *   state symbols  *  EleCmdMix3.htm  * working out formulae  *


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.

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'.

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).


diagram of paper/thin layer chromatography at start diagram of paper/thin layer chromatography at the end diagram of paper/thin layer chromatography at the end diagram of paper/thin layer chromatography at the end

(a) At the start mark a baseline in pencil - 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.

(b) The prepared chromatogram paper is carefully placed in the solvent so the baseline and spots are above the solvent. The solvent is allowed to move up the paper. The colours begin to separate out. Its 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.

(d) How do we measure an Rf value? Its quite simple to measure a reference value (Rf) value 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 larger glass container with a lid on, it reduces evaporation of the solvent.

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

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 Rf values below and diagrams above). The Rf values is the ratio how far the spot travels (D on diagram) relative to the distance moved by the solvent (S on the diagram) and must have values of >0 and <1.

   distance moved by substance spot (D)
Rf  = --------------------------------------------------------------
   distance moved by solvent front (S)

Reference values Rf = distance move by substance

Chromatography Rf 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.

THEORY of thin layer/paper chromatography

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 solubilities 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.

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

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

However, if one dye is more strongly held by the paper (stationary phase), its progress up the paper by dissolving in the solvent 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 are all in the food dye (6), the dye being analysed.

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!

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 Rf value and has a value of 0.0 (not moved - no good), to 1.0 (too soluble - no good either), but Rf ratio values between 0.1 and 0.9 can be useful for analysis and identification of coloured dye-like materials.

Rf = distance moved by dissolved substance (solute) / distance moved by solvent.

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 and coloured purple by a chemical reagent called Ninhydrin.

(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 Rf 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, 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 described below

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.



  • Gas-liquid chromatography (gas chromatography, gc/glc/g.l.c.) can be used to analyse liquid mixtures which can be vapourised (e.g. petrol, blood for alcohol content). The instrument is called a gas chromatograph.

    • a picture of 'glc': diagram a gas chromatogram and the resulting chromatograph

    • A sample of the substance under investigation is injected and vapourised into a tube containing a carrier gas (called the mobile phase, it moves). The mobile phase must be an inert or unreactive gas like nitrogen.

    • The gas carries the vaporised substance through a long 'separating' tube or column wound around inside a thermostated oven. Inside the column is the immobile phase (stationary phase) which is usually a thick viscous liquid/porous solid which has some affinity with the substances to be analysed in the carrier gas.

    • The substances in the mixture are partially absorbed by an absorbent material held in the or column (called the immobile phase or stationary phase, which doesn't move), but only temporarily. However different substances are held back, or 'retained', for different times so that the mixture separates out in the carrier gas stream.

      • There is a dynamic equilibrium between the stationary and mobile phases and the separation of the components of a mixture by chromatography depends on the distribution of the components in the sample between the mobile and stationary phases.

      • The more a substance is strongly held by intermolecular forces, the slower that component moves through the separating column (stationary phase). Therefore, depending on the different force of attraction, the components get separated out and emerge from the column into the detector at different times.

      • The column is filled with a porous solid so gas can get through but passes over a large surface area OR it is coated in a very high boiling organic liquid which can also provide a large absorbing surface but still allows gas flow.

    • The gases emerge from the oven into a detector system which electronically records the different signal as each substance comes through.

      • A printout or computer display of the results from the gas chromatograph, called the gas chromatogram, shows a series of peaks in the graph line imposed on a steady baseline when only the carrier gas is passing through the detector.

      • The time it takes for a substance to come through is called the retention time and is unique for each substance for a particular set of conditions (flow rate, length of separating column, nature of separating column material, temperature etc.).

    • Generally speaking, the greater the molecular mass of the mixture molecule, the longer the retention time.

      • This is because the component molecule - immobile phase intermolecular force of attraction increases with the size of the component molecule, so it is absorbed/retained temporarily a bit more strongly (see right of diagram).

    • The height of the peak, or more strictly speaking, the area under the peak, is proportional to the amount of that particular substance in the mixture.

    • Therefore it is possible to both identify components in a mixture and calculate their relative proportions.

    • The chromatogram shown above (right of chromatograph diagram) illustrates the separation of some alkane hydrocarbons in petrol (in reality it is far more complicated with dozens of hydrocarbon molecule peaks on the chromatogram). The different peak heights give the relative proportions i.e. hexane >pentane > heptane.

    • The retention time order follows the trend of increasing molecular mass gives increasing retention time i.e. in time heptane C7H16 > C6H14 > C5H12

    • The gas chromatographic instrument can be calibrated with known amounts of known substances.

      • So, the timing position of the peak identifies the component X in the gas and the height of the peak tells you much of X is in the mixture.

      • Note - if there is only one peak, only one substance has been detected. This may mean its a pure substance, or a substance containing molecules that cannot be detected by the particular gas chromatograph system.


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