Chromatography Of Chlorophyll, Of Amino Acids: Experiment Process And Results

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Chromatography is the process of separating a mixture. Chromatography is used for a varied amount of applications and industrial processes such as for the pharmaceutical industry, food industry, and in many other processes. In the Pharmaceutical industry it may be used for the separation of chiral compounds. Pharmaceutical companies also like to use it to prepare very large amounts of extremely pure materials, and to analyse the purified compounds for some trace contaminants. In the food industry chromatography may be used to control a product’s quality by separating and analysing vitamins, proteins, preservatives and amino acids. Different types of chromatography include Gas chromatography, Thin layer Chromatography, Liquid Chromatography and Paper Chromatography. Chromatography can tell you many things about solvents and mixtures such as the number of components in the mixture, sometimes the identity of compounds, and the purity of a compound. Chromatography happens in two different stages, the mobile phase and the stationary phase the stationary phase does not move during the process however the mobile phase moves along the page with the sample, the mobile phase is a compound use to separate components in mixture, whereas the stationary phase is where materials to be separated are selectively adsorbed. Adsorption is defined as the process where a solid holds molecule of solids, gas’ or liquids or solute as a thin film. Chromatography works when we have the mixture moving over the surface of something else in another state of matter (depending on what state the mixture started in) that stays stationary. The substance that moves is called the mobile phase and the substance that remains in one place is called the stationary phase. As the mobile phase is moving, it separates into individual components onto the stationary phase, we can then identify them. Factors that affect Chromatography include polarity and molecule size etc, with polarity, knowing the polarity of the chosen solvents helps you get an indication of how quickly or slowly the components in the mixture move up the stationary phase and separate out. Solvents that are more polar allow the components in the mixture to move quicker than substances that are less polar. The aim of Chromatography is to understand a substance, therefore Rf values are calculated at the end of the practical’s, Rf values can tell you what compound an unknown material is if it is the same as a known substance.

Chromatography of Chlorophyll

Aim of the investigation:

Break down Chlorophyll in a leaf to see what colours you will find

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Method:

Equipment list:

Pipette, Capillary tube, Pestle and Mortar, boiling tube, Bung or Cling film, heating system (hair dryer), pencil, Chromatography paper, Ruler, Propanone, Pigment sample (In this case holly and Spinach leaf). Dissection scissors and Sellotape.

  1. Finely cut provided Holly leaves or Spinach leaves using the dissection scissors and pace the cut pieces into a mortar.
  2. Add sand to the mortar and an appropriate amount of Propanone to make the leaves grind easier and to ensure the leaves are broken properly so liquid is released, and cell membranes are broken to ensure chlorophyll and other substances are present.
  3. Grind the mixture until there is enough liquid to carry out the required amounts of drops on each piece of chromatography.
  4. Make sure that filter paper or Chromatography paper is cut to fit into the boiling tube, and label each of the three pieces of paper using pencil and in the corner of the paper write how many drops of the crushed leaf you will add e.g. 1, 3 and 6.
  5. Draw the pencil line on each piece of the Chromatography paper at least one cm from the bottom
  6. Add the appropriate number of drops to each piece of the chromatography paper using a small amount of the sample each time by picking it up with a capillary tube, making sure that the drop is dried in between using a hairdryer, so concentration builds up.
  7. Pour 3cm cubed of propanone in the boiling tube and start the chromatography process by placing in the samples making sure the pencil line does not touch the solvent.
  8. Add cling film to the top of each of the three boiling tubes and wait until the solvents reach at least one inch from the top of the paper.
  9. Calculate Rf values

Results Analysis and Evaluation

  • 1 spot- 1 (10.7/10.7)
  • 3 spots- 0.77 (6.5/8.4)
  • 6 spots- 0.92 (7.9/8.5)

Overall the results were pretty unexpected apart from the anomaly from the 1 spot, before the investigation it was predicted that 6 spots would give the highest Rf value because it would be more concentrated so it would separate more, and it was predicted that 1 spot would have the lowest Rf value and separate the least because it was much less concentrated than the other two dots. From looking at the chromatogram the results for our 1 dot sample were strange, the line was basically invisible up until 8.5 cm where there was a small section of green pigment which then turned into a tan orange colour for about 1 cm. This high Rf value means that the spot and the solvent line travelled together (which they did very rapidly) normally a high Rf value like this also means that the substance is very polar, however spinach is not and the chlorophyll in it is not extremely polar, this leads me to believe that the sample taken for dot one contained a lot propanone than the rest did making the spot more polar than the rest. This means that the investigation would have worked much better if we controlled the distribution of propanone and spinach, which we will try our best to do in our next attempt at the practical with the TLC plate.

Another fact which could have affected the practical was at times we found that we had to dip the capillary tube in the mortar where the spinach was as we would pick up more of the leaf than the actual liquid, this meant we may have had again uneven convention drops, for instance the dots with less liquid we’re likely to not contain a lot of Chlorophyll meaning there’s likely to be less pigmentation and clear separation causing inconsistent results and Rf values. If pieces of leaf did end up onto the chromatography paper, this could have also affected the results as this may have blocked or limited separation.

Part of the investigation which did go well was the set up, the chromatograms in all three beakers were watched closely during lesson time and all three were set up the same, this includes paper length and width as-well as the amount of solvent used

Chromatography of Amino acids

Aim of investigation:

understand substances in the ER mixture and compare the differences in the amino acids.

Method:

Equipment:

DL LEUCIC, YSINE HCL, ASPARTIC ACID, ER MIXTURE, Watch glass, Beaker, Ethanoic acid, Butan-1-oil, Distilled water, Ninhydrin, capillary tube, hairdryer, chromatography paper, Fume cupboard, pencil, glasses.

Firstly, 3ml of water, 6ml of Butan-1-ol and 1.5ml of Ethanoic acid was poured into a beaker and a watch glass was placed over the beaker to saturate the air.

  1. A large strip of chromatography paper was sectioned into 4, each section was about 2cm wide with a 1cm gap in between each section to prevent possible spreading of cross contamination of the different amino acids
  2. Prepare the chromatography paper by drawing a line 1.5cm form the bottom of the paper using a pencil
  3. Each section of the paper was labelled with each of the amino acids at the very top ensuring that it would not encounter the later solvent line or the components.
  4. A capillary tube was then used to apply the amino acids to the correctly labelled chromatography paper, one small dot was placed on each
  5. Each single dog was then dried out using a hairdryer, ensuring that the hairdryer was not held too close to the amino acid sample to prevent denaturing enzymes
  6. Steps 4and 5 were repeated for another 2 dots
  7. the samples were then carefully placed into the beaker making sure that the solvent does not touch the pencil line.
  8. the watch glass was placed back on top of the beaker and the beaker was placed in a fine cupboard
  9. The solvent was left in the fine cupboard until the solvent line travelled to at least 1.5cm away from the top of the paper.
  10. the chromatography paper was taken out the fine cupboard by a teacher and sprayed with ninhydrin to make the amino acids visible
  11. results were analysed appropriately and Rf values were calculated

Results analysis and evaluation

The two results from the ER mixture and DL-LEUCIC we’re generally like the rest of the class’ result, this also makes sense or the results in our group also because these two amino acids are less polar. Also form research, many websites had similar ranges with the Rf value (normally in a 0.1-15 range), so clearly this part of the practical was done well.

The Rf values (rounded)

  • DL LEUCIC- 0.87
  • LYSINE HCL- 0.1
  • Aspartic acid- 0.13
  • ER MIXTURE-0.87

From comparing my groups result to the practical to the rest of the group’s results, we could see that are Rf values for Lysine HCL and Aspartic acid were much lower than expected. This could be because over-concentration of a spot, uneven movement of the solvent in the TLC or excessive spot sizes. It’s also understood that very polar molecules such as these two amino acids tend to not travel very far up the chromatography paper because they interact more with the stationary phase. As the stationary phase is polar (more polar compounds like the Aspartic acid and Lysine, these tend to have a lower Rf value.

Over spotting may have also been a very likely cause of these anomalies, as for the most part of the practical we could not see the amino acids once they dried on the paper, meaning that the dot distribution may have been uneven or over concentrated which causes unexpected results.

Tilting seems not to be a problem here because we set up the chromatography so it was straight and attached to the watch glass lid, also the chromatogram was underneath close surveillance as we watched our slow rising solvent lines in the fume cupboard, and not once did it move, so this part of the practical also went particularly well.

Chromatography of Chlorophyll on TLC plate

Aim of investigation:

To compare the results with the previous attempt, evaluate and consider which works best.

Method:

Equipment list:

  • Spinach
  • Watercress
  • Pestle and mortar
  • Propane
  • Capillary tube
  • Beaker
  • Chromatography solvents (5 parts cycto hexane, 3 parts pyopanone and 2 parts hexane
  • Hairdryer
  • Goggles
  1. Finely cut spinach and water cress we’re added to the pestle and mortar and we’re grounded until it produced liquid, propanol was added to help liquify.
  2. The TLC plate was prepared by drawing a pencil line 1.5 cm for the bottom of the plate.
  3. The chromatography solvent was added to the beaker, around 2 cm was added to the beaker and the air was that saturated by putting a watch glass on top
  4. The ground up spinach and other leaf was then picked up by the capillary tube and one small dot was placed on the TLC plate
  5. Step 4 was repeated but before each dot the previous dot was dried using a hairdryer
  6. The TLC plate was placed in the beaker and the watch glass was placed back on the top of it
  7. Wait and leave the TLC plate in the beaker until the solvent line reached approximately 1.5 cm form the top of the plate
  8. Results were analysed and separated and Rf values were calculated

Results analysis and Evaluation

The results for the TLC plate clearly showed that the experiment had worked better with the plate than with the paper, on the TLC much more pigment had shown through so we could see more colour had come out of the spinach leaf the reason for this is that the intermolecular forces between the paper and the pigment is very weak however with the TLC plate the intermolecular forces are very strong between the paper and pigment.

  • Rf value 0.95 (rounded)

The Rf value in this investigation was very close to 1, from research I found that this meant that the substance used which in this case is water cress and spinach mix simply means that they’re very non-polar, so they move rapidly with the solvent. The high Rf values could also be a result of poor technique, for example uneven movement of the solvent in the TLC plate, excessive spot size or an over concentrated spot. Another reason why this may have happened may be because of the positioning of the TLC plate, the plate was tilted in the beaker, although it should have been flat against the beaker floor. From looking at the images of the practical, the spotting of the spinach and cress looks fine, the spot looks no bigger than 1mm-1.5mm in diameter and the separation looks more like separation rather than streaking form the dot.

The results from were generally like the rest of the class’ result, we all received a much more clear and higher results from the previous attempt using filter/chromatography paper. However this is debatable as we do not know whether this more pigmented and better result is due to the TLC plate or the addition of the water cress, this is an improvement that could have been made, to truly see which one of the two stationary phases works better with just spinach. Generally TLC plates work better than paper chromatography because it is a known trend that TLC plates show greater separation of the components, which makes analysis of the results much easier especially when it came down to seeing where new colours were forming.

References

  1. Science.jrank. Industrial applications of Chromatography (online) Available at: (Accessed 13 November 2019)
  2. Study read. Types of chromatography (online) Available at: (Accessed 20 November 2019)
  3. Sciencing- Why does chromatography work? (online) Available at : (Accessed 1st January 2020)
  4. Differences between mobile and stationary phase (online) Available at: (Accessed 3rd January 2020)
  5. Stationary phase definition (online) Available at: Accessed 3rd January 2020)
  6. Chromatography (online) by Chris Woodford Available at: (Accessed 3rd January 2020)

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