SEPERATE TO IDENTIFY:
CHROMATOGRAPHY
1. INTRODUCTION AND THE BASICS 3. CALCULATIONS 5. EVALUATION OF TECH
PLANT PIGMENTS PAPER CHROMATOGRPAHY
INTRODUCTION TO CHROMATOGRPAHY CALCULATING RETENTION FACTORS A large majority of is
RF stands for retention factor which is the ratio between the distance moved by the sample from the origin line to the distance moved practical related to pla
At the beginning of the chromatography practical, we set
What is chromatography? Chromatography is a technique Plant pigments are light absorbing-coloured by the solvent front. RF values give a numerical value to the distance the samples have travelled up the chromatography plate. The with the solvent. Due
out to produce both a paper and thin layer chromatogram
used to separate a mixture into its separate components. macromolecules. The majority of a plant’s pigments are !"#$%&'( )*+(, -. #%)/0(
correctly place the chr
equation used to calculate the Rf values is 𝑅𝑓 = . The measurements need to be taken from the starting line to of plant pigments however, this did not happen. When
chlorophyll; chlorophyll absorbs violet-blue light and !"#$%&'( )*+(, -. #*0+(&$
There are two phases within the chromatography process, first time. During a pr
the centre of each separated substance, the solvent front is taken from the starting line up to the furthest point the solvent has travelled. trying to place the prepared paper into the solvent the chromatography, the T
the stationary phase and the mobile phase. The stationary reflects green light, giving plants their green colour.
Chlorophyll is found within chloroplasts (where The RF value must always have a value between 0 and 1. Our solvent front was calculated at 48 mm and the final TLC chromatogram insubstantial nature of the paper meant that it could not resulted in the solvent
phase is usually a porous solid” (Britannica, 2021) that is
photosynthesis takes place), the chlorophyll is there to can be seen in figure 3.1. self-stand in the beaker and ultimately meant the solvent then meant that the se
used to facilitate the separation of the liquid. The mobile
surpassed the origin line and submerged the pigment have been. Additional
phase is a solvent, liquid or gas that carries the sample up absorb the violet-blue light needed for photosynthesis to
occur. There are two classifications of chlorophyll, 9 sample. Since the sample was submerged, we did not beaker, we should hav
through the stationary phase for separation. The stationary Pigment 1, Light Green 𝑅𝑓 = = 0.19 instantaneously to pre
chlorophyll a, and chlorophyll b. They differ slightly in 48 obtain an accurate separation of the plant pigment and
and mobile phases facilitate a mixture being separated evaporating. When co
could not calculate any precise Rf values. The one Rf
into its individual components. The mixture's overall structure, so they absorb slightly different wavelength of 12 53 forgotten to place the
separation depends on how “strongly attracted the light; this maximises the total amount of visible light Pigment 2, Dark Green 𝑅𝑓 = = 0.25 value that we did calculate was = 1, showing that the
34 Solvent 53 minutes later; this cou
chemicals are to the mobile and the stationary phases” absorbed and makes photosynthesis more effective. As well Pigment 4 pigment stopped travelling at the same point as the chromatogram.
as the main chlorophyll pigments, plants also have accessory 14 Front
(BBC Bitesize, 2021). The mixture this is being separated Pigment 3, Purple 𝑅𝑓 = = 0.38 solvent front and was most likely a technical error and not
pigments. Accessory pigments help absorb the remaining 34
can be from a vast range of substances including amino a correct result. Because the results were not accurate and
acids, plant pigments, inks and others. There are multiple wavelengths of light not absorbed by the chlorophyll to the techniques needed drastically changing, we decided to
optimise the rate of photosynthesis of the plant. The main 46 Pigment 3
forms of chromatography including thin layer; paper; gas; Pigment 4, Yellow 𝑅𝑓 = = 0.96 not re-run the paper chromatography due to time
column; and high-performance liquid chromatography. accessory pigments found in plant leaves are carotenoids, 48 Pigment 2 constraints. The paper chromatogram we obtained can be
Our investigation consisted of the separation of plant anthocyanins and xanthophylls. Chlorophyll b can also seen in figure 5.1.
pigments using a solvent, thin layer chromatography and sometimes be classified as an accessory pigment as is in Origin Pigment 1
paper chromatography. paucity in comparison with chlorophyll a. line
Figure 3.1 – TLC Chromatogram
4. ANALYSIS OF CHROMATOGRAMS 6. SUGGESTED IMPROV
2. METHOD COMPARISON OF RF VALUES SIZE OF THE MOLECULES
Once the Rf values have been calculated we can determine what As well as the polarity of the plant pigment molecules, their size will affect
pigments are present within our plant sample by comparing our Rf values the distance that they travel up the chromatogram. The larger the molecule There were multiple factors that we could have changed throughout the practi
THE EQUIPMENT USED TO CARRY OUT THIS THIN LAYER CHROMATOGRPAHY to standard Rf values of plant pigments. Using the table seen in figure the less mobile it is and so it will not travel as far up the chromatogram. data we collected. For example, if we had access to a longer TLC plate, we co
PRACTICAL ARE AS FOLLOWS: 4.1, we can speculate that pigment 1 is chlorophyll b, pigment 2 is Oppositely, the smaller the pigment molecule the further it will travel as it is a longer amount of time. This would have facilitated the better separation of th
more mobile. Comparing the plant pigment structures seen in figure 4.2 to
• Plant leaves (our plant was a tradescantia zebrina plant seen in figure 2.2) chlorophyll a, pigment 3 is anthocyanin and pigment 4 is carotene. The their distance travelled on our chromatogram we can see links between size provided more accurate Rf values when we were calculating. Additionally, to
• Acetone Rf values calculated from our chromatography practical fit well into the of the molecule and the distance travelled. Given that chlorophyll a and calculations we could have marked the exact middle of each pigment before a
• A watch glass standard Rf values, while also taking into consideration that not all plants chlorophyll b are extremely similar in structure it makes sense that they are When drying the solvent could further lifted the pigments up the chromatogram
• Capillary tubes will provide the same values. relatively close on the chromatogram. Moreover, they are both considerably
• 100cm3 beakers large molecules compares to the anthocyanin and carotene and so they not as distance travelled which is then needed to calculate the Rf values. As previou
• Fine grain sand mobile and thus didn’t travel a large distance while the solvent was running. conduct a paper chromatogram and did not carry out a re-run due to time cons
The third furthest up the chromatogram is the anthocyanin. From figure 4 we
• Pencil
can see that it is substantially smaller than both chlorophyll molecules as so
chromatography practical’s, we could use a boiling tube and a cork cut in half
• Ruler it could travel further as it is more mobile. Lastly, the carotene travelled solvent is running. By assisting the paper from entering the solvent on an angl
• TLC plates furthest up the chromatogram and so must be smaller than both the would have gained far more accurate results and could have seen better separa
• Pestle and mortar chlorophyll molecules and the anthocyanin molecules. This also mirrors the
• Solvent (a mixture of cyclohexane, propanone and petroleum ether) calculate Rf values.
idea that carotene is the most polar and chlorophyll b is the least given the
• A heater/hair dryer distance that they travelled up the chromatogram.
• Pipette
POLARITY
The equipment used can be seen in figure 2.1
Polarity is when one end of a molecule has a slightly negative charge, and the
TO CONDUCT THIS PRACTICAL USE THE FOLLOWING METHOD: other end has a slightly positive charge. Polarity is caused when there is an
1. Place 1-2 leaves into the mortar and crush using the pestle; this may be aided with a few grains of fine sand “accumulation of electron density at one end of the molecule” (ChemPages,
2. Add 1ml of acetone to the mortar using a pipette to create an extract
3.
4.
Collect the supernatant from the mortar and place a thin layer onto the watch glass and try to cover a large surface area
Dry the layer of extract using a heater to evaporate the water
2021) which causes the slight negative and positive charges. Due to the
charges in the molecules, polar solvents and polar molecules will be more THE STATIONARY PHASE 7. REFERENCES
strongly attracted to one another than a polar solvent and non-polar molecule. While the basic method of conducting a chromatography practical is the
5. Repeat steps 3 and 4 to create a concentrated amount of supernatant on the watch glass The solvent used in the thin layer chromatography was a polar solvent. W ith same for both paper and thin layer chromatography, they have contrasting
6. Re-hydrate the extract using a few drops of acetone this in mind, we can determine that whichever pigment travelled furthest up properties that make mixtures run differently. The stationary phase in paper Britannica. (2021) Stationary Phase. [online] Available at:
https://www.britannica.com/science/stationary-phase-chromatography [Accessed 26 Apr. ChemPages. (2021). Molecular Pol
7. Draw a pencil line 1cm from the bottom of the TLC plate; ensuring a light pressure is used the chromatogram is the most polar molecule. Therefore, the pigment that ran chromatography contains cellulose, a polymer of the simple sugar glucose; 2021]. https://www2.chem.wisc.edu/deptfil
8. Using a capillary tube pick up some of the extract and dot it onto the pencil line the lowest up the chromatogram must have a lower polarity. In the case of our due to the -OH groups in the polymer chain, the paper/stationary phase is rces/01review/review5.htm [Access
Supriya N (2019) Difference Between Paper and Thin Layer. [online] Available at:
9. Dry the dot using a heater, then place more of the extract onto the same dot and dry again; repeat 5 times to ensure a chromatogram, carotene travelled the further so must be more polar and polar. Because the -OH groups make the cellulose very polar they are
https://biologyreader.com/difference-between-paper-and-thin-layer-chromatography.html Nold, M. (2018). Chlorophyll a and
concentrated amount of plant pigment is on the TLC plate chlorophyll b travelled the least distance so is must be less polar. However, we strongly attracted to polar water molecules, even water vapour in the air. [Accessed 30 Apr. 2021]. Available at: https://www.sigmaaldr
must also consider the size of the pigment molecule, as this will affect the Therefore, the surface of the paper is coated in water molecules that make reporter/analytical-standards-for-nat
10. Pour 0.5cm3 depth of solvent into the 100cm3 beaker and place the chromatogram into the solvent. Making sure that the solvent the paper polar and strongly attracted to both the solvent (in this case water) Tarragó-Celada, J. Novell, J. (2019) Colour, chlorophyll and chromatography. [online]
does not directly touch the spot/is above the pencil line distance it can travel up the chromatogram. Additionally, the stationary phase Available at: https://www.scienceinschool.org/content/colour-chlorophyll-and- WOM. (2021). The B-Carotene Mo
can also be polar which can create an attraction between the stationary and and the plant pigments. As for thin-layer chromatography, the stationary chromatography [Accessed 30 Apr. 2021].
11. Place a lid over the beaker and allow the solvent to run for 40 minutes phase is comprised of a glass plate with a thin layer of silica gel (this could
at: https://www.worldofmolecules.c
mobile phases. In our chromatography practical we conducted thin layer
12. Once the solvent has travelled two thirds of the way up the TLC plate, remove it from the beaker and mark the solvent front chromatography and paper chromatography were both stationary phases and
also be aluminium oxide); silica is a type of silicon dioxide. The surface of BBC Bitesize. (2021) Chromatography. [online] Available at: Valavanidis, A. and Vlachogianni, T
https://www.bbc.co.uk/bitesize/guides/ztkdd2p/revision/1 [Accessed 30 Apr. 2021].
with a pencil the silica gel also contains -OH groups, which means that the stationary Chemistry, [online] pp.269–295. Av
mobile phases were polar which may have improved the separation of the https://www.sciencedirect.com/scien
13. Allow to dry and calculate Rf values pigments. phase is very polar. Attraction to the stationary phase is present for both the SAPS. (2015) TLC of plant photosynthetic pigments. [online] Available at:
https://www.saps.org.uk/secondary/teaching-resources/1347-a-level-set-practials-tlc
paper and thin layer chromatography and therefore, it cannot be a reason for
[Accessed 1 May 2021].
the unsuccessful paper chromatogram.
