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CHEMISTRY : Chromatography


RELEASE DATE: 30th Dec 98 - 23rd Feb 99
LAST UPDATE: 2 Sep 2009
VERSION HISTORY: 1.0 - First release
1.1 - ? (23 Feb 1999)
1.2 - Revision of text and formatting. (2-Sep-2009)


Recentely, I was interested in seperating and perhaps isolating the different dyes used particularly in inks, such as those used in pens. For such a purpose the technique used is Chromatography. However, a standard method must be deviced, so that all chromatography tests and Analysis are done by the same standard routine. Due limiting factors of my home lab, the method of choice is Paper chromatography, where the components making the dye/ink are seperated by solvent on a special chromatography paper medium.


Method 1 Diagram

The first method (shown in picture above) was simply done by immersing a strip of chromatography paper inside a measuring cylinder. This was held from the top by a match or rod perforated through the strip and placed on the cylinder mouth as shown. Only the wide mouth measuring cylinder was adequate as the smaller ones were not practical to use, especially when the wet paper used to stick with the sides of the cylinder. Also since I had only one wide diameter measuring cylinder. not more than one chromatography test could be performed. Another disadvantage was that the plastic cylinder was not good for certain solvents such as Chloroform and Terachloromethane which are examples of plastic solvents.


Method 2 Diagram

Side-sticking effect was a big problem and was avoided by eliminating the use of a closed cylinder around the chromatography paper. This method (shown in picture above) consisted of suspended chromatography strips in air over petri dishes or culture plates (used in microbiology for media) filled with the solvent. The strips were suspended by sticking them to a horizontal rod which was attatched with strings to a support above. This was very easy to run 4 or 5 tests together side by side, and eliminating the sticking-side effect.

Unfortunately the plastic culture plates were dissolved in chloroform and interferrd negatively, since dissolved plastic absorbed by the Chromatograohy paper slowed and stopped the flow. But the major disadvantage was that since everything was exposed to air, the solvents evaporated rapidly (especially in hot summer days!). It evaporated both from the the plates and the paper itself, and this intereferred with the experiment. For this reason a closed system was inevitable and the following method was invented.


Since I had quite some disposable MSU bottles from the bacteriology Lab, I decided that I could use these to make a closed environment for the chromatography paper strip. One MSU bottle (10cm long) was by far 2 short. Unfortunately, the plastic with which was made the MSU bottle did dissolve with some solvents such as tetrachloromethane and chloroform - but this problem is tackled in an adaptation discussed below.
Two MSU bottles over each other were hence used to form a cylinder with the required length where the the base of the upper tube was cut off. The 2 tubes were then joined together with super glue (or chloroform which melted the plastic). Instead of suspending the chromatography strip by the attatched rod (as in method 2), the screw tap of the MSU bottle was perforated with a rectangular thin slit, through which the chromatography paper strip pass through until it meets the solvent layer at the base. It is kept freely suspended in the solvent (that is not resting at the base of the tube) by either using an adhesive tape between one end of the strip, and the tap itself, or better still by using a pressed straw in the slit of the cap which presses against the edge of the slot and paper strip and keeps it in place. The latter had an advantage that the strip could be easy lowered or raised so that it would be in a suitable place in the solvent.
Since of a closed and relatively narrow tube, the side-sticking effect was again a problem. However I came with the idea that if I insert 2 metal needles or thin rods next (horizontally parallelel) to each other right through the MSU bottle wall somewhere at the lower part of the tube, spaced about 6-8mm apart and then pass the chromatography strip in between the needles, it will prevent the paper from moving and hence not adhere with the sides of the tube.
The needles are easily inserted and fixed in the plastic bottle by heating on a flame to red hot and perforate the plastic bottle using a pair of pliers. The needle parts which are out of the tube were cut with the pliers, the holes around the needles are sealed with super glue, and the site of perforation is covered with a plastic tape.
For plastic solvents, a long wide bore test tube is used, using the same slit-perforated screw described above. The side sticking effect can be avoided if the strip is straight and immersed slowly. Later, I bought special type of stationary metal clips. These were clipped at the bpttom of the chromatography paper strip and immersed in thesolvent. The weight provided by the strips at the base of the paper improved greatly the situation regarding side-sticking effect. This provided excellent chromatography results. A diagram of this method is found below in the procedure section.


Preparation of the Chromatography paper strip

  1. A 15cm by 1cm chromatography stip was cut.
  2. A horizontal line 1cm from one end is drawn with a pencil. This is the application base (origin) where the drop of ink/test material is applied.
  3. 10 markings, 1.2 cm each are drawn above each other along any of the strip sides, thus giving a total length of 12cm. The extra space above is used to to label the strip, and from where it could be attatched with the tap above. Each of the markings represent 10% of the length of solvent absorbed since that a full length of 12cm is used.
A diagram of how the setup should look like is shown in the picture below:

Method 3 Diagram

The Chromatography procedure

  1. A small drop of ink (or just scribble a large 'fullstop' if a pen is used) is placed on the centre of the application line. Place in a clean place.
  2. About 2.0 -2,5 mls of solvent is transferred at the base of the MSU bottle or glass test tube using a long neck glass pipette. Avoid wetting the sides of the tube with solvent.
  3. The chromatography strip is now inserted from the tap slot and hold from falling inside by inserting a straw in the slot. The straw's outward pressure will hold the strip in place against the edge of the slot edge
  4. The strip is lowered through the needles and further down till its end is about 2mm below the solvent superficial layer. If timing is performed, start recording time as the solvent reaches the material ink drop under test.
  5. This is left till the solvent rises at least 70% of the length. If two or more colour seems to seperate, it is recommended to leave till 90 - 100% for wider separation. If the solvent seems that it has stopped flowing(no change in 5 mins) the strip should be removed.
  6. This is left to dry in air, prefeebbly by suspending vertically in air

Isolation of the Dye(s) in solution

  1. If seperated materials need to be isolated, then the zones with dye(s) are cut out from the chromatography paper by a pair of scissors.
  2. These paper are placed in 5-10ml solvent (eg ethanol). Not more than 10mls solvent should be used so as to obtain a rather saturated solution of the dye(s).
  3. When the paper is clear from the dye (hence dissolved in the solution) it is removed using a metal tweezers
  4. The solution can be concentrated by letting to evaporate in room temperature. A fastest way is to apply heat some distance above an electric flame (eg. over a hot plate.) NEVER HEAT FLAMMABLE MATERIAL DIRECTLY OVER NAKED FLAMES


Plastic solvents like Chloroform used to dissolve the plastic container and interefered with te chromatography tests. An adaptation of the above method was used so that all types of organic solvents can be used. The alteration had of course to eliminate the plastic container (MSU bottles) and replaced with a wide bore (2cm diameter) glass test tubes.

The chromatography strip length, markings, screw cap with rectangular slit + straw is identical to the method above. To keep the strip vertically central and not touch the inside wall of the glass test tube, a mass is added to the base of the strip. It is immersed in the solvent which often is inert to metal objects. A set of uncoated stainless steel clips (which can be easily bought from any stationary) were used for this purpose. If only coated ones can be fbought, the plastic coating can be removed by aceone which act as a paint remover. Another excellent item used as weight at the base of the paper were 1cm wide metal clips, though these may not be easily found for sale. Any other weights that do not react with solvent are can be used , for example small magnet pieces at opposite sides of the paper strip.
A diagram of this is shown below:

Method 4 Diagram


  1. Quite easy to perform - 'dip and go!'
  2. Sticking side effect avoided
  3. Closed system, no evaporation of (toxic) solvents
  4. Several tests can run together using different solvents (great for comparative analyis)
  5. Little solvent is required
  6. All types of solvents can be used.
  7. Long chromatography strip (12-15cm) allows adequate separation, depending solvent


  1. The only disadvantage, like any paper chromatography method, is that the test take a lot of time - typically 45 to 90 minutes.
  2. This method also yield little amount of pigments when it comes to the extraction of the isolated pigments. Several tests must be run to gather a respectable amount for further chemical tests.


  1. When I stuck the two MSU bottles with chloroform (instead super glue) I moved them near the hot plate so that heat evaporate the chloroform and solidify faster. This caused hot chloroform vapour turn the plastic opaque, and nothing could be seen.
  2. Diluting the solvents could improve the separation properties. Separation score of 50% ethanol = 9/10, while that of 100% ethanol = 1/9 !!! ) Read solvent pages for more info. (Click here).
  3. Do not leave tests unvigilated for a very long time. Fast solvents tend to push all dyes to a line at the top of the strip.
  4. Before using an unknown / new solvent, check wether it dissolves plastic or not.
  5. Always do markings, labeling and any writing on the chromatography paper with a pencil. Inks will dissolve in the solvent and interfere.

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