The investigation of Chlorophyll using Thin Layer Chromatography

Updated: Oct 23, 2019


Chromatography, is a well known method used to separate compounds using two phases; a mobile as well as a stationary phase. In Thin Layer Chromatography, the stationary phase is a thin layer such as silica on a plate and the mobile depends on the sample that is investigated. The sample is spotted on the origin line of the TLC plate and put upright in a TLC chamber with eluent in the bottom, in which the capillary action develops. The TLC plate is removed before the eluent reaches the top and the calculation of the retardation factor or Rf value is obtained. Using the value obtained and comparing it with a known value, one can for example identify the compound at hand. Generally a known and an unknown compound are investigated to identify the unknown one.

What led me to gain interest and want to investigate Chlorophyll extracted spinach using TLC, is the use of it in the forensic field, used to identify and compare drugs, explosives, inks and dyes at crime scenes. The only thing that limits the use of TLC, is the need of the samples to be soluble, which can be limiting to investigate e.g. pen inks with gel pens that are insoluble.

However, pros to TLC are how colourless compounds can be investigated using fluorescence and UV-light to find the dark spots or spraying the TLC plate with a chemical such as ninhydrin that reacts with the amino

acids of a compound to bring about colours, mainly purple

and brown to the compound.

It really interests me, the use of chemistry, how with analyzing polarities and Retardation Factor values one can solve mysteries that might not be visible to the naked eye. Specially intriguing how, to understand compounds on a molecular level gives a better understanding of the chemical and physical properties on a macroscopic level.

Background information

Thin layer chromatography, being one of the most modern methods of separating mixtures in chemistry, utilizes a mobile phase and a stationary phases. Mobile phase is a solvent that carries the sample and the stationary phase on the TLC plate. The stationary phase, also known as the adsorbent, is a solid polarity like surface bound to the TLC plate and certain well known example for a stationary phase is Silica (SiO2), which will be used to perform the investigation.

Silica is a polar substance, due to the surface crystals consisting of polar hydroxyl (OH) groups, the mobile phase will be an an organic solvent mixture and the solvent, a relatively non-polar substance in contrast to the silica adsorbent. As the sample mixture (in this case extracted Chlorophyll ) is applied to a small spot by the origin of the TLC plate with a capillary also known as “The process of spotting”, the plate is then put into a TLC chamber for the solvent to rise up the plate and the Chlorophyll will dissolve into the mobile phase known as “Developing the TLC plate”.

Before the eluent reached the top of the TLC plate, it should be removed, Solvent front marked and the Rf value should be calculated using the following formula:

The value can then be compared with a known one to identify compounds, measure polarity, affinity and analyze a compound in whole. During the development of the TLC plate, the components are separated based upon polarity. Polar molecules, generally tend to spend a greater amount of time holding onto the polar stationary phase than non-polar molecules. The stronger the molecules clings to the stationary phase, the slower will it the mobile phase carry it across the plate. Thus, due to different affinities and polarities of compounds tested, results differ.

Chlorophyll, with a porphyrin ring structure, is the molecule found in the chloroplast of the plants that are responsible for the green colour of the leaves. They are capable of absorbing specific wavelengths of visible light and then convert them into chemical energy to perform photosynthesis. In plants, one can find two different types of Chlorophyll , Chlorophyll A and Chlorophyll B, which will be isolated from spinach leaves for this investigation.

The most abundant Chlorophyll is Chlorophyll A, although many plants contain a significant amount of Chlorophyll B with a different of an aldehyde group at the C-7 position rather than a methyl group in Chlorophyll A,

making B more polar than A.

Their are also other pigments other than Chlorophyll such as “Carotenoids” that leave a bright yellow, orange and red tint on the leaves during fall, due to Chlorophyll degrading to a colourless compound during that time. Thus, once the pigments from the spinach are extracted, a complex mixture of Chlorophyll and carotenoids are left which are separated using chromatography as well.

Carotenoids are a larger collection of plant-derived compound with the name “Terpenes”. They are naturally occurring compounds, and considering they contain either 10, 15, 20, 25, 30 and 40 carbon atoms, it means that they have a compound with a 5 carbon atom chain that serves as their building block. The “head to tail” bonding fashion making their isoprene units join together which forms terpenes and this is known as the isoprene rule. The branches end is the head and the branches is the tale. Carotenoids are tetrapernes and an example being β-carotene, responsible for the orange colour in carrots.

Spinach leaves, mainly contain Chlorophyll A & B and β-carotene as their major pigments, but their are other smaller compounds such as α-carotene, Xanthophylls and Pheophytin that are oxidized versions of Carotenes. The α- and β-carotene differ only in their position of double bond in the outermost cyclohexene rings. But the rest of the carotene consist of methyl groups (-CH3) as well as conjugated polyenes (alternating single and double bonds). Why Carotene is considered the least polar compound in the bunch, is because its most polar functional group are the methyl groups and no other.

Pheophytin is a grey pigment related to Chlorophyll, with the difference of that its Porphyrin ring does not have Magnesium ion in the center, but two different protons instead. However, like Chlorophyll, it has Porphyrin A with a methyl group and Porphyrin B with a aldehyde group, making B more polar than A and thus A not really visible as well. It is due to the differences in the porphyrin central atoms of Chlorophyll and Porphyrin that makes Chlorophyll more polar than Porphyrin.

Xanthophylls with a yellow pigment is the most polar dye in contrast to the others, due to their ability to exhibit Van der waals, dipole-dipole and hydrogen bonding interactions. Just like the Carotenes, it has conjugated polyenes and methyl groups as well as alcohol groups located at each end.

As a conclusion, the different components all have different visible colours, which can be observed and thus their separation on the TLC paper can be followed visually. When it comes to polarity, the β-carotene is a hydrocarbon and thus is very non-polar. But the Chlorophylls contain C---O and C---N bonds being polar groups and Magnesium bonded to Nitrogen all form an overall strong polar bond. Meaning, both Chlorophylls are more polar than β-carotene. Between the Chlorophylls, Chlorophyll B has a aldehyde group (-CHO) where Chlorophyll A has a methyl group (-CH3), making B more polar than A.

The hypothetically best eluent from the available ones in school according to the background information above, the solvent that is most focused on and of which a polar gradient will be made of as well is Hexane. Hexane is very non-polar and thus tested purely and also with other polar solvents such as Ethyl Acetate and Methanol giving a polar gradient that can help with the polarity analysis.

Research question

A further and more explanatory research question for this investigation would be;

  • The investigation of different eluents in Thin layer chromatography on Chlorophyll Retardation Factor value and polarity.


Hexane is the main focus, considering the TLC plate has Silica Gel on it which is very polar and thus a non-polar mobile phase should be used to get the best Retardation Factor value as possible. Also carotene is the least polar among the rest of the compounds and so if a polar eluent would be used, all the compounds would be washed down and that is not the aim of this investigation. Thus it is logical to use eluents according to their polarity and the order of polarities amongst the different compounds I predict are; Hexane on Carotene and Pheophytin, Ethyl Acetate on Chlorophyll and lastly Methanol on the most polar being Xanthophylls.

However, if the following chosen eluents dont work, I will try more to further analyze my investigation. In general considering I am only investigating the extracted chlorophyll and not each dye individually, I predict that the sample will move most with the mobile phase for the non-polar compound Hexane and also in the polar gradients with Hexane having the highest ratio in the mixture. Meaning, the more polar the eluent mixture gets, the less should the sample of Chlorophyll move up from the origin.

Method and apparatus

Following chemicals were used;

Spinach, Methanol CH4O relative polarity; 0.762, Ethanol C2H6O relative polarity; 0.654, Ethyl acetate C4H8O2 relative polarity; 0.228, Hexane C6H14 relative polarity; 0.009, 1-propanol C3H8O relative polarity; 0.617, 2-propanol C3H8O relative polarity; 0.546, Butan-1-ol C4H10O relative polarity; 0.586, 2-Butanone C4H8O relative polarity; 0.327, Hydrochloric acid HCL relative polarity; quite high.

Following apparatus were used:

Sizzer, TLC paper & silica gel, Tweezer, Pipettes, Pen, Ruler ±0.05 cm, Gloves, 3 beakers, Measuring cylinder ±0.1 ml, Parafilm, Mortar, Stove

Extraction of Chlorophyll from spinach:

(1) Using a mortar, few spinach leaves were put inside and smashed to pieces.

(2) 50 ml of rubbing alcohol (such as ethanol) was put inside the mortar and further blended until the solution got an even green colour.

(3) Using a coffee filter, the solution was filtered into a beaker, leaving the leaves behind and put on a stove.

(4) The solution boiled for a few minutes until a concentrated Chlorophyll solution was left with a dark green colour.

(5) Another coffee filter was used to make sure no residue leaf was inside the solution into yet another beaker.

(6) The Chlorophyll solution was left to cool and then was ready to use.

Preparing the eluent solutions:

(1) Many different eluents were tested as well as a few mixed together to analyse the outcome and expand the trials.

(2) Each eluent would have its own pipette and small measuring cylinder.

(3) If it would be a mixed solution, they would all be measured in a measuring cylinder using a pipette and then poured into a beaker to be mixed before use.

List of eluents used are the following:

  • Methanol

  • Ethanol

  • Hexane

  • Ethyl Acetate

  • Hexane : Ethyl Acetate : Ethanol (in different ratios listed in the table in analysis section)

  • Hexane : Ethyl Acetate : Ethanol : Methanol (in different ratios listed in the table in analysis section)

  • 2-propanol : Buta-1-non

  • 1-propanol : Hexane

  • 1-Butanol

Thin layer chromatography process:

(1) TLC silica gel paper was handled with gloves and cut 4cm wide and 10cm long with a ruler and pen as the origin line.

(2) A thin line was drawn 1cm from the base of the paper carefully and an X was drawn in the middle of the line.

(3) A small capillary was used to extract the Chlorophyll and place on to the X mark and the spot was dried with a hair dryer.

(4) Step 3 was repeated a minimum of 3 times to make sure the spot was concentrated and packed with Chlorophyll.

(5) Using a tweeter and gloves, the TLC paper was carefully picked up and placed inside a beaker with eluent solution (called the TLC chamber) with the eluent under the origin line.

(6) A paradigm was quickly cut and placed on top of the beaker in order to make sure no evaporation takes place and the process can go smoothly.

(7) After a few minutes the Chlorophyll spots had moved up and ready to be removed from the chamber and the front line was quickly drawn with a pen before getting dry.

(8) Each eluent was tried out 3 times in 3 different beakers In order to get an average Rf value for the analysis.


There are no particular safety issues when performing this investigation. However, when working with pouring the eluents into the beaker to perform TLC, it should be done in a fume hood to avoid too much air contamination. Also, the TLC paper needs to handled with a lot of care using gloves to not damage its surface. However, the chemicals used as solvents to perform TLC are mostly irritating to the skin, flammable as well as harmful to the human body and nature. Thus they should also be handled with care to not get in contact with the skin, be inhaled much and also disposed of in a waste beaker and given to the teacher to be taken care of in an environmentally friendly manner.

Result and analysis

Collected data;


According to my Hypothesis, I believed that whenever a non-polar eluent such as the Hexane would be used to perform the TLC, or when the non-polar compound was at highest ratio in a polar gradient mixture, the Chlorophyll sample would move up the most and when a polar solvent would be used, it would move the least. My prediction compared to my data, shows that my hypothesis was wrong and instead the more polar eluents seemed to move up the sample the most.

With the help of the tables in the result and analysis section as well as the picture to the right, the TLC procedure with only pure Hexane showed a seperation of green, dark green and yellow as well as yellow line right by the solvent front. In fact, Pure Hexane and methanol were the only eluents that showed a visual separation of compounds, which after the investigation of multiple different trials with different eluents makes the assumption questionable again.

With pure Hexane, a yellow colour formed around the fang-like green shape of the sample. Not only did the sample not move up even with it having the lowest relative polarity in comparison to the other eluents, but the sides stretched and only a small yellow line reaching the solvent front. The colour separations make me assume that the separations were the green colour

“Chlorophyll A”, the dark green colour “Chlorophyll B” and the yellow “β-carotene”.

With methanol, a yellow circle separated from the sample and the green circle went all the way up to the solvent front. Meaning, the yellow colour was less polar and thus moved less than the green and so I believe the yellow colour was in fact β-carotene.

From the processed data section in table 11, one can clearly see Hexane having the lowest Rf value. In general, a high Rf value, means a compound has high polarity and a low Rf value means the compound is nonpolar, which means my data on Retardation values have quit a large limitation.

Ethyl Acetate also gave quit a good result when it comes to separating Chlorophyll A and B on one of the trials. However, the separation turned into a horizontal thick line of dark green and light green dye. As a whole, Ethyl acetate, Hexane and Methanol gave the best results with separations. So for the next time, I would most likely want to keep my focus on mainly Ethyl Acetate, Methanol and Hexane to experiment them separately again as well as in gradient mixtures with different ratios trying to keep Hexane as the highest ratio but to also try with the others as a high ratio.

Table 4 & 8 present all the collected information in regards to the TLC trials performed using 1-butanol and Propan-1-ol. From visual observation, these two eluents gave me no proper Rf values, considering many of the trials ended up as streaks of lines and also the compounds in a gradient mixture didn't seem to help much either.

Ethanol as an eluent alone did also give many results with only streaks and also didn't seem to contribute positively in a gradient mixture. Several gradient mixtures were tested without Ethanol added into them, and no particular difference was visually notices.

Thus, I believe for the future, Butanol, Propanol and Ethanol should be eliminated from future trials and in future investigations. If possible, to get a hold on a more polar eluent or several other non polar eluents, much more analysis will be investigated.

Evaluation and further investigation

Considering some of the TLC results, “Uneven advanced samples” and “Streaking” occured, which can be improved in the following ways.

Uneven advanced samples occurred due to the sides of the TLC paper touching the sides of the TLC chamber. Getting the sample crooked or uneven makes it harder to measure the Retardation factor, considering it becomes more difficult to know how far the sample has traveled or where the middle is. By getting a bigger chamber for the TLC paper to just tilt on, would help it out and to be even more careful with the TLC paper for the silica layer to not fall off or get damaged.

Streaking samples occurred in one of my trials working with methanol as my eluent, which also makes it harder to know how far the sample traveled or where the middle is. Several reasons behind such a reaction exists, which can be improved. One being to make sure not to overload the sample on the origin line, which might have been the case considering I added the sample and dried it multiple times before performing the reaction. Another improvement could be that the solvent systems polarity might be damaged and thus a more appropriate one should be applied. The last reason could be the different compounds in the sample itself affecting the polarity difference and thus interrupting the travel up the TLC paper.

As mentioned earlier in the conclusion section, the Rf values seem to show quite a large limitation. The probability of the reason behind it being the measurement with the ruler is very low considering the rulers uncertainty as well as calculated uncertainties were very low. Thus the probability of it being because of the eluents, the extracted chlorophyll as well is TLC papers is higher.

The problem might lie behind the extracted chlorophyll that the method was correct, but the process of high heat and particular alcohol might have damaged the remaining concentrated chlorophyll after all the alcohol was evaporated. So, for the next time, a ready made professionally extracted chlorophyll could improve the current investigation to an extent. But if not possible, another method should be used instead without the need of heat. Chlorophyll extraction through evaporation overnight or over several days might give a better result. But, then the extracted Chlorophyll will then have to be already prepared before the investigation.

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