Chromatography Of Chlorophyll and Amino Acids: Experiment Process And Results

Chromatography is a technique used to separate components of a mixture, finding widespread application across various industries, including pharmaceuticals, food processing, and more. In the pharmaceutical sector, it separates chiral compounds, prepares high-purity materials, and detects trace contaminants in purified compounds. The food industry employs chromatography to ensure product quality by identifying and quantifying vitamins, proteins, preservatives, and amino acids.

There are several chromatography methods, such as Gas Chromatography, Thin Layer Chromatography, Liquid Chromatography, and Paper Chromatography. These techniques can reveal the number of components in a mixture, identify compounds, and assess a compound's purity.

Chromatography operates through two phases: the mobile phase and the stationary phase. The stationary phase remains fixed, while the mobile phase carries the sample across the stationary phase, facilitating the separation of mixture components based on selective adsorption. Adsorption refers to the adhesion of molecules from a gas, liquid, or dissolved solid to a surface, creating a thin film.

The effectiveness of chromatography hinges on the interaction between the mobile and stationary phases. As the mobile phase moves, it distributes individual components onto the stationary phase for subsequent identification. Key factors influencing chromatography include the polarity and size of molecules. For instance, the polarity of solvents can determine the speed at which mixture components migrate through the stationary phase, with more polar solvents typically accelerating this movement.

The ultimate goal of chromatography is to analyze and understand substances. This is often achieved by calculating Retention Factor (Rf) values at the end of an experiment. Rf values can help identify unknown substances by comparing them to known compounds, offering insights into the composition and purity of the analyzed materials.

Investigation of Chlorophyll Chromatography

Objective: To dissect chlorophyll from a leaf and identify the spectrum of colors it contains.

Equipment Required:

  • Pipette and Capillary tube
  • Pestle and Mortar
  • Boiling tube, Bung or Cling Film
  • Hair Dryer (as a heating system)
  • Pencil and Ruler
  • Chromatography Paper
  • Propanone (Solvent)
  • Pigment Sample (Holly and Spinach Leaves)
  • Dissection Scissors and Sellotape


  1. Finely chop the provided Holly or Spinach leaves using dissection scissors and place them into a mortar.
  2. Introduce sand into the mortar along with a suitable quantity of Propanone. This facilitates easier grinding of the leaves, ensuring thorough breakdown of the leaves to release liquid and break cell membranes, thus liberating chlorophyll and other substances.
  3. Grind the mixture until enough liquid is produced for the experiment.
  4. Prepare the chromatography paper by cutting it to fit inside the boiling tube. Label three pieces of paper with a pencil, indicating the number of pigment drops to be applied (e.g., 1, 3, 6).
  5. Draw a baseline one cm above the bottom edge of each chromatography paper strip.
  6. Apply the designated number of drops from the leaf extract onto each strip using a capillary tube. Ensure each drop is thoroughly dried with a hairdryer before applying the next to increase concentration.
  7. Pour three cm³ of Propanone into the boiling tube and initiate the chromatography by inserting the paper strips, ensuring the pencil line remains above the solvent level.
  8. Seal the top of each boiling tube with cling film and allow the solvent to ascend to at least one inch from the top of the paper.
  9. Upon completion, calculate the pigments' Retention Factor (Rf) values.

This systematic approach aims to reveal the diverse color components present within chlorophyll, offering insights into its complex composition.

Analysis and Evaluation of Results

  • Single Spot: Rf = 1 (10.7/10.7)
  • Three Spots: Rf = 0.77 (6.5/8.4)
  • Six Spots: Rf = 0.92 (7.9/8.5)

The outcomes of this experiment were largely unforeseen, except for the single spot's result, which deviated from initial predictions. The six-spot sample was anticipated to yield the highest Rf value due to its higher concentration, presumably leading to more effective separation. Conversely, the single spot was expected to exhibit the lowest Rf value and most minor separation due to its lower concentration than the other samples.

Upon examining the chromatogram, the results for the single-spot sample were peculiar. The chromatography line remained nearly invisible up to 8.5 cm, where a brief segment of green pigment transitioned into a tan-orange hue over approximately 1 cm. This unexpectedly high Rf value suggests that the spot and the solvent progressed rapidly. Typically, a high Rf value indicates a highly polar substance. However, given that spinach and its chlorophyll content are not exceedingly polar, it's plausible that the single-spot sample was disproportionately saturated with Propanone, rendering it more polar than the others.

This observation implies that the experiment could have benefited from more precise control over the distribution of Propanone and spinach. In future iterations of this practical exercise, particularly with Thin Layer Chromatography (TLC) plates, efforts will be made to standardize the distribution of Propanone and spinach to achieve more consistent and reliable results.

Another factor that influenced the experiment's outcomes was the method of collecting the sample with the capillary tube and, occasionally, dipping the tube into the mortar containing the spinach resulted in picking up more leaf material than liquid. This could lead to uneven distribution of the samples, where dots with less liquid might not have contained sufficient chlorophyll, leading to weaker pigmentation and less distinct separation. This variability could contribute to inconsistent results and Rf values. Furthermore, if leaf fragments were transferred onto the chromatography paper, they could obstruct or hinder the separation process, further impacting the accuracy of the results.

However, a positive aspect of the experiment was the consistency in the setup across all trials. The chromatograms for all three samples were meticulously monitored throughout the class, ensuring uniformity in paper dimensions and the volume of solvent used. This standardized approach across the setups helped maintain control over the experiment, allowing for a more reliable comparison of the results from each sample.

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Chromatography of Amino Acids

Objective of the Investigation: To analyze the components within an ER mixture and discern the differences among various amino acids.

Equipment Required:

  • DL Leucine, Lysine HCl, Aspartic Acid, ER Mixture
  • Watch Glass, Beaker
  • Ethanoic Acid, Butan-1-ol
  • Distilled Water, Ninhydrin Solution
  • Capillary Tube, Hairdryer
  • Chromatography Paper
  • Fume Cupboard
  • Pencil, Protective Glasses


  1. Combine 3ml of water, 6ml of Butan-1-ol, and 1.5ml of Ethanoic Acid in a beaker. Cover with a watch glass to saturate the air with the solvent mixture.
  2. Cut a large strip of chromatography paper into four sections, each approximately 2cm wide, ensuring a 1cm gap between sections to prevent cross-contamination.
  3. Using a pencil, draw a baseline of 1.5cm from the bottom of the chromatography paper.
  4. Label each section's top with the amino acids' names, ensuring labels are placed well above the solvent line and sample application area.
  5. A capillary tube is used to apply a small dot of each amino acid to its corresponding section on the paper.
  6. Dry each applied dot with a hairdryer, avoiding overheating and potentially denaturing the amino acids.
  7. Repeat steps 5 and 6 to apply three dots per amino acid.
  8. Carefully insert the paper into the beaker, ensuring the solvent does not touch the pencil-drawn baseline.
  9. Cover the beaker with the watch glass again and place it within a fume cupboard.
  10. Allow the solvent to ascend the paper until it reaches 1.5cm from the top, ensuring consistent chromatogram development.
  11. Remove the chromatography paper from the fume cupboard under supervision and spray with ninhydrin to visualize the amino acids.
  12. Analyze the results and calculate each amino acid's Retention Factor (Rf) values.

This method facilitates identifying and comparing amino acids within an ER mixture, providing insights into their distinct properties and behaviors during chromatographic separation.

Analysis and Evaluation of Results

The ER mixture and DL-Leucine outcomes from our experiment aligned closely with those of the rest of the class, which was anticipated, given that these two amino acids exhibit lower polarity. Research from various sources also indicated similar Rf value ranges (typically between 0.1 and 0.15), suggesting this experiment segment was conducted effectively.

Rf Values (Rounded):

  • DL Leucine: 0.87
  • Lysine HCl: 0.1
  • Aspartic Acid: 0.13
  • ER Mixture: 0.87

Upon comparing our group's results with those of the entire class, it was observed that our Rf values for Lysine HCl and Aspartic Acid were notably lower than expected. Potential reasons for this discrepancy could include:

  • Over-concentration of the sample.
  • Inconsistent solvent movement on the TLC plate.
  • Overly large spot sizes.

It is recognized that highly polar molecules, such as Lysine HCl and Aspartic Acid, tend to migrate less on the chromatography paper due to more robust interactions with the polar stationary phase, resulting in lower Rf values.

Another possible factor contributing to these anomalies could be over-spotting. Given the difficulty in visualizing the amino acids once they had dried on the paper, there's a chance that the application of the samples could have been more balanced and more concentrated, leading to skewed results.

The chromatography setup was error-free, as the apparatus was positioned correctly and secured to the watch glass lid. Continuous monitoring of the solvent's progression in the fume cupboard confirmed that the chromatography paper remained stable throughout the experiment, eliminating tilting as a potential issue.

Overall, while certain aspects of the practical were executed successfully, the variations in Rf values for Lysine HCl and Aspartic Acid highlight areas for improvement, particularly in sample application techniques and ensuring uniform solvent movement.

Chromatography of Chlorophyll on TLC Plate

Objective: To assess and compare the effectiveness of this chromatography method with previous attempts to determine the optimal approach.

Equipment Required:

  • Spinach and Watercress
  • Pestle and Mortar
  • Propanol
  • Capillary Tube
  • Beaker
  • Chromatography Solvents (5 parts Cyclohexane, three parts Propanone, two parts Hexane)
  • Hairdryer
  • Protective Goggles


  1. Finely chop the spinach and watercress, then grind them in the pestle and mortar until a liquid is produced. Add propanol to facilitate the extraction process.
  2. Using a pencil, prepare the TLC plate by drawing a baseline 1.5 cm from the bottom edge.
  3. Mix the chromatography solvents and pour about 2 cm into a beaker. Cover with a watch glass to saturate the air with solvent vapors.
  4. A capillary tube is used to apply a small dot of the ground leaf mixture onto the marked TLC plate.
  5. After applying each dot, use a hairdryer to dry it before adding the next, ensuring each application is thoroughly dried.
  6. Place the TLC plate into the beaker and re-cover with the watch glass.
  7. Allow the TLC plate to remain in the beaker until the solvent front is approximately 1.5 cm from the top of the plate.
  8. Once complete, analyze the results, identify the separated components, and calculate the Rf values.

Conclusion: This experiment aims to refine the chromatography process by comparing the results with previous attempts, thereby identifying the most effective method for analyzing chlorophyll on a TLC plate.

Analysis and Evaluation of Results

The chromatography experiment utilizing a TLC plate demonstrated a marked improvement over previous attempts with paper chromatography. The TLC plate revealed significantly more pigment from the spinach leaf, indicating a more effective separation. This enhanced performance is attributed to the more vital intermolecular forces between the TLC plate and the pigments compared to those between paper and pigments.

Rf Value: 0.95 (rounded)

The Rf value obtained in this experiment was notably close to 1, suggesting that the watercress and spinach mixture substances are highly non-polar, allowing them to move swiftly alongside the solvent. However, the high Rf value might also reflect methodological flaws, such as uneven solvent movement on the TLC plate, overly large or concentrated spots, or improper positioning of the TLC plate, which was observed to be tilted rather than flat.

Upon review of the practical images, applying the spinach and watercress mixture appeared appropriate, with spot sizes between 1mm and 1.5mm in diameter, indicating effective separation rather than streaking from the spot.

The results aligned with the rest of the class, showing more precise and pronounced outcomes than those obtained using filter or chromatography paper. Whether the improved results are attributable to using the TLC plate or the inclusion of watercress remains to be seen. Further experimentation is necessary to determine which stationary phase—TLC plate or paper—yields better results with spinach alone. Generally, TLC plates are recognized for providing superior component separation over paper chromatography, facilitating more accessible analysis, and identifying new colors' emergence.


  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 1 January 2020)
  4. Differences between mobile and stationary phase (online) Available at: (Accessed 3 January 2020)
  5. Stationary phase definition (online) Available at: Accessed 3 January 2020)
  6. Chromatography (online) by Chris Woodford Available at: (Accessed 3 January 2020)

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